U.S. patent application number 12/089292 was filed with the patent office on 2009-12-10 for plants having an increased content of amino sugars.
This patent application is currently assigned to Bayer CropScience AG. Invention is credited to Bernd Essigmann, Claus Frohberg.
Application Number | 20090304889 12/089292 |
Document ID | / |
Family ID | 37906527 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090304889 |
Kind Code |
A1 |
Frohberg; Claus ; et
al. |
December 10, 2009 |
Plants having an increased content of amino sugars
Abstract
The present invention relates to plant cells and plants having
an increased content of N-acetylated glucosamine derivatives.
Furthermore, the present invention relates to plant cells and
plants which synthesize glucosaminoglycans. The present invention
also provides processes for producing said plants and compositions
comprising said plant cells.
Inventors: |
Frohberg; Claus;
(Kleinmachnow, DE) ; Essigmann; Bernd; (Berlin,
DE) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W., SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Bayer CropScience AG
Monheim am Rhein
DE
|
Family ID: |
37906527 |
Appl. No.: |
12/089292 |
Filed: |
October 5, 2006 |
PCT Filed: |
October 5, 2006 |
PCT NO: |
PCT/EP06/09776 |
371 Date: |
September 18, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60725388 |
Oct 11, 2005 |
|
|
|
Current U.S.
Class: |
426/518 ;
435/419; 536/128; 800/278; 800/284; 800/298 |
Current CPC
Class: |
C12N 9/1096 20130101;
C12N 15/8246 20130101; C12N 15/8243 20130101 |
Class at
Publication: |
426/518 ;
800/298; 800/278; 800/284; 435/419; 536/128 |
International
Class: |
A23P 1/00 20060101
A23P001/00; A01H 5/00 20060101 A01H005/00; C12N 15/82 20060101
C12N015/82; C12N 5/10 20060101 C12N005/10; C07H 1/08 20060101
C07H001/08 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2005 |
EP |
05090279.0 |
Sep 22, 2006 |
EP |
06090177.4 |
Claims
1. A plant cell or plant comprising a content of N-acetylated
glucosamine derivatives of at least 2 .mu.mol per gram of fresh
weight.
2. A plant cell or plant comprising a content of glucosaminoglycans
of at least 300 .mu.g of glucosaminoglycan per gram of fresh
weight.
3. A part of the plant of claim 1, wherein said part of the plant
comprises a content of N-acetylated glucosamine derivatives of at
least 2 .mu.mol per gram of fresh weight.
4. A part of the plant of claim 2, wherein said part of the plant
comprises a content of glucosaminoglycans of at least 300 .mu.g of
glucosaminoglycan per gram of fresh weight.
5. Propagation material of the plant of claim 1, wherein said
propagation material comprises a content of N-acetylated
glucosamine derivatives of at least 2 .mu.mol per gram of fresh
weight.
6. Propagation material of the plant of claim 2, wherein said
propagation material comprises a content of glucosaminoglycan of at
least 300 .mu.g of glucosaminoglycan per gram of fresh weight.
7. A process for producing a genetically modified plant comprising:
a) introducing a foreign nucleic acid molecule coding for a protein
having the activity of a glutamine:fructose 6-phosphate
amidotransferase of isoform II (GFAT-2) or coding for a protein
having the activity of a bacterial glutamine:fructose 6-phosphate
amidotransferase into a plant cell; b) regenerating a plant from
plant cells obtained according to step a) c) optionally, generating
further plants with the aid of the plants according to step b).
8. A process for producing a plant which synthesizes
glucosaminoglycan comprising a) genetically modifying a plant cell
comprising steps i to ii in any order or carrying out any
combinations of the following steps i to ii individually or
simultaneously i) introducing a foreign nucleic acid molecule
coding for a protein having the activity of a glutamine:fructose
6-phosphate amidotransferase of isoform II (GFAT-2) or coding for a
protein having the activity of a bacterial glutamine:fructose
6-phosphate amidotransferase (bacterial GFAT) into a plant cell ii)
introducing a foreign nucleic acid molecule coding for a
glucosaminoglycan synthase into a plant cell b) regenerating a
plant from a plant cell comprising the genetic modification
according to steps i) a) i ii a) ii iii) a) i and a) ii, c)
introducing into plant cells of plant according to step i) b) i a
genetic modification according to step a) ii, ii) b) ii a genetic
modification according to step a) i, and regenerating a plant d)
optionally, generating further plants with the aid of the plants
obtained according to any of steps b) iii or c) i or c) ii.
9. (canceled)
10. A process for producing glucosaminoglycans comprising the
extraction of glucosaminoglycans from the plants cell claim 2.
11. A composition comprising a genetically modified plant cell of
claim 1.
12. (canceled)
13. A process for producing flour comprising grinding the part of a
plant of claim 3.
14. (canceled)
15. A process for producing glucosaminoglycans comprising the
extraction of glucosaminoglycans from the part of a plant of claim
4.
16. A process for producing glucosaminoglycans comprising the
extraction of glucosaminoglycans from the propagation material of
claim 6.
17. A composition comprising a genetically modified plant cell of
claim 2.
18. A process for producing flour comprising grinding the part of a
plant of claim 4.
Description
[0001] The present invention relates to plant cells and plants
having an increased content of N-acetylated glucosamine
derivatives. Furthermore, the present invention relates to plant
cells and plants synthesizing glucosaminoglycans. The present
invention also provides processes for producing said plants and
compositions comprising said plant cells.
[0002] The amino sugar glucosamine, glucosamine derivatives and
polymers comprising glucosamine derivatives are used, inter alia,
as food supplements for the prophylaxis of joint disorders in
animals and man. In the medical field, too, some glucosamine
derivative-containing polymers are used for treating disorders.
[0003] WO 06 032538 describes transgenic plants which had been
transformed with nucleic acid molecules coding for hyaluronan
synthases. The synthesis of hyaluronan in the plants in question
could be demonstrated unambiguously.
[0004] WO 98 35047 (U.S. Pat. No. 6,444,878) describes a metabolic
path for the synthesis of GlcNAc in plant cells where glucosamine
is converted by a number of successive enzymatically catalyzed
reaction steps with formation of the metabolites GlcNAc,
N-acetylglucosamine 6-phosphate, N-acetylglucosamine 1-phosphate
into UDP-GlcNAc. A metabolic path which was described as an
alternative for plants comprises the conversion of fructose
6-phosphate and glutamine into glucosamine 6-phosphate which is
then converted by a number of successive enzymatically catalyzed
reaction steps with formation of the metabolites glucosamine
1-phosphate and N-acetylglucosamine 1-phosphate into UDP-GlcNAc.
The conversion of fructose 6-phosphate and glutamine into
glucosamine 6-phosphate is catalyzed by a protein having the
activity of a glutamine:fructose 6-phosphate amidotransferase
(GFAT) (Mayer et al., 1968, Plant Physiol. 43, 1097-1107).
Relatively high concentrations of glucosamine 6-phosphate are toxic
for plant cells (WO 98 35047).
[0005] WO 00 11192 describes the endosperm-specific overexpression
of a nucleic acid molecule from corn coding for a protein having
the enzymatic activity of a plant GFAT in transgenic corn plants
with the aim of synthesizing a cationic starch having
2-amidoanhydroglucose molecules in plants. The metabolic path
described which, according to the description of WO 00 11192,
should result in the incorporation of 2-aminoanhydroglucose into
the starch, comprises inter alia the incorporation of
UDP-glucosamine by starch and/or glycogen synthases into the
starch. It was possible to demonstrate increased amounts of
UDP-glucosamine in the flour of endosperm of the transgenic corn
plants in question overexpressing a nucleic acid molecule coding
for a protein having the enzymatic activity of a plant GFAT
translationally fused with a plastid signal peptide. When the
protein having the enzymatic activity of a GFAT was expressed
without signal peptide, it was possible to demonstrate an increased
amount of glucosamine 1-phosphate in the corresponding flour from
corn endosperm tissue. It was not possible to detect cationic
starch or increased amounts of N-acetylated glucosamine
derivatives, such as, for example, UDP-GlcNAc or
N-acetylglucosamine 6-phosphate, in the transgenic plants.
[0006] The amino sugar beta-D-glucosamine (glucosamine) and/or
derivatives of glucosamine are components of various polymers
(glucosaminoglycans) which, inter alia, are essential components of
the exoskeleton of arthropods, the extracellular matrix of mammals
or the exopolysaccharides of some bacterial microorganisms.
[0007] Thus, for example, N-acetyl-D-glucos-2-amine
(N-acetylglucosamine, GlcNAc) is a glucosamine derivative
acetylated at the nitrogen atom. GlcNAc is, for example, a
molecular building block of hyaluronan (beta-1,4-[glucuronic acid
beta-1,3-GlcNAc].sub.n), which is an essential component of the
synovial fluid.
[0008] In the medical field, hyaluronan-containing products are
currently used for the intra-articular treatment of arthrosis and
as ophthalmics used for eye surgery. Derivatized cross-linked
hyaluronan is used for treating joint disorders (Fong Chong et al.,
2005, Appl Microbiol Biotechnol 66, 341-351). In addition,
hyaluronan is a component of some rhinologics which, for example in
the form of eye drops and nasalia, serve to moisten dry mucous
membranes. Hyaluronan-containing solutions for injection are used
as analgesics and antirheumatics. Patches comprising hyaluronan or
derivatized hyaluronan are employed in wound healing. As dermatics,
hyaluronan-containing gel implants are used for correcting skin
deformations in plastic surgery. In cosmetic surgery, hyaluronan
preparations are among the suitable skin filler materials. By
injecting hyaluronan, for a limited period of time, it is possible
to smooth wrinkles or to increase the volume of lips.
[0009] In cosmetic products, in particular in skin creams and
lotions, hyalauronan is frequently used as a moisturizer by virtue
of its high water-binding capacity. Furthermore,
hyaluronan-containing preparations are sold as so-called
neutraceuticals (food supplements) which can also be used in
animals (for example dogs, horses) for the prophylaxis and
alleviation of arthrosis.
[0010] The catalysis of the hyaluronan synthesis is effected by a
single membrane-integrated or membrane-associated enzyme, i.e.
hyaluronan synthase (DeAngelis, 1999, CMLS, Cellular and Molecular
Life Sciences 56, 670-682). Hyaluronan synthase catalyzes the
synthesis of hyaluronan from the substrates UDP-glucuronic acid
(UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc).
[0011] Hyaluronan used for commercial purposes is currently
isolated from animal tissues (roostercombs) or prepared
fermentatively using bacterial cultures.
[0012] Proteoglycans, a class of glycoproteins, are, inter alia, an
essential component of cartilage and have, attached to a core
protein, glucosaminoglycans composed of repetitive disaccharide
units. The repetitive disaccharide units for their part are
covalently attached to the core protein via a characteristic
carbohydrate binding sequence. Depending on the composition of the
disaccharide units, a distinction is made, inter alia, of the
glucosaminoglycans heparan/heparin sulfate, keratan sulfate and
chondroitin/dermatan sulfate, whose disaccharide units each contain
a molecule which is either glucosamine or a glucosamine derivative.
In these substances, sulfate groups are introduced at various atoms
or substituents of the disaccharide units, so that the respective
substances mentioned are not uniform polymers but polymer groups
summarized under the respective generic term. Here, the individual
molecules of the polymer groups in question may differ both in the
degree of sulfation and in the position of the monomers containing
sulfate groups.
[0013] The synthesis of the disaccharide chain of the
chondroitin/dermatan ([beta-1,4]-[glucuronic acid
beta-1,4-N-acetylgalactosamine].sub.n) is catalyzed by a
chondroitin synthase starting with UDP-GlcA and
UDP-N-acetylgalactosamine, an epimer of UDP-GlcNAc (Kitagawa et
al., 2001, J Biol Chem 276(42), 38721-38726). The glucuronic acid
molecules of chondroitin can be converted by an epimerase into
iduronic acid. If more than 10% of the glucuronic acid molecules
are present as iduronic acid, the polymer is referred to as
dermatan. The introduction of the sulfate groups in various
positions of the disaccharide chain of the chondroitin or the
dermatan is then catalyzed by further enzymes, resulting in
chondroitin/dermatan sulfate. Here, the degree of sulfation may
differ from molecule to molecule.
[0014] For some time, chondroitin sulfate has been considered as a
potential active compound for treatment of osteoarthritis (Clegg et
al., 2006, The New England Journal of Medicine 354(8),
795-808).
[0015] The synthesis of the disaccharide chain of heparin/heparan
(heparosan) ([alpha-1,4]-[glucuronic acid
beta-1,4-glucosamine].sub.n or [alpha-1,4]-[iduronic acid
alpha-1,4-glucosamine].sub.n) is catalyzed by a heparin/heparosan
synthase from UDP-GlcA and UDP-GlcNAc (DeAngelis und White, 2004,
J. Bacteriology 186(24), 8529-8532). The glucuronic acid molecules
of the heparin/heparosan can be converted by an epimerase into
iduronic acid. The introduction of the sulfate groups in various
positions of the disaccharide chain of the heparosan is then
catalyzed by further enzymes, giving rise to heparin sulfate or
heparan sulfate. Heparin sulfate has a considerably higher
substitution by sulfate groups than heparan sulfate. Heparin
sulfate has about 90% iduronic acid molecules, whereas in the case
of heparan sulfate the fraction of glucuronic acid molecules
predominates (Gallagher et al., 1992, Int. J. Biochem 24, 553-560).
As in the case of chondroitin/dermatan sulfate, in the case of
heparin/heparan sulfate, too, the degree of sulfation may differ
from molecule to molecule.
[0016] Heparin sulfate is used, inter alia, as an anticoagulant,
for example for the prophylaxis and treatment of thromboses.
[0017] Chondroitin/dermatan sulfate and heparin/heparan sulfate are
currently produced by isolation from animal tissues. Chondroitin
sulfate is mainly isolated from bovine or shark cartilage, and
heparin/heparan sulfate is isolated from porcine intestine or
bovine lungs. Since the disaccharide chains of chondroitin/dermatan
sulfate or heparin/heparan sulfate have no uniform sulfation
pattern, it is difficult to obtain a uniform specific product.
Accordingly, the products are always mixtures of molecules with
varying degrees of sulfation.
[0018] The glucosaminoglycan chitin ([beta-1,4-GlcNAc].sub.n) is
one of the main components of the cell wall of fungi and the
exoskeleton of insects, millipedes, arachnids and crustaceans and
is a polymer which is insoluble in water. The enzyme chitin
synthase catalyzes the synthesis of chitin by linking UDP-GlcNAc
(Merzendorfer and Zimoch, 2003, J. Experimental Biology 206,
4393-4412).
[0019] As a raw material source for isolating chitin, use is to
date mainly made of crustaceans (prawns, crabs) and fungi, such as,
for example, Aspergillus spec., Penicillium spec. Mucor spec. WO 03
031435 describes, for example, a method for preparing GlcNAc by
fermentation of yeasts. Depending on the method by which chitin is
isolated from the raw material source in question, chitin contains
in addition to GlcNAc also its deacetylated form glucosamine as a
building block. If more than 50% of the building blocks are GlcNAc,
the polymer is referred to as chitin, whereas polymers comprising
more than 50% of glucosamine are referred to as chitosan. These
days, glucosamine or derivatives thereof, such as, for example,
GlcNAc, are produced by degradation of chitin. Chitin may either be
deacetylated first, resulting in the formation of chitosan, or be
degraded directly, resulting in the formation of GlcNAc.
[0020] Chitin can be deacetylated enzymatically with the aid of
chitin deacetylases (Kafetzopoulos et al., 1993, Pro. Natl. Acad.
Sci. 90, 2564-2568) or by chemical deacetylation.
[0021] The degradation of chitin or of chitosan can also take place
both enzymatically (for example using chitinases, glucanases,
beta-N-acetylglucosaminidases), and by chemical hydrolysis.
[0022] The degradation of chitosan or the deacetylation of GlcNAc
results in the formation of glucosamine.
[0023] A substantial disadvantage of all methods for preparing
amino sugars by degradation of chitin consists in the fact that,
owing to incomplete hydrolysis and/or incomplete deacetylation,
what is obtained is not a uniform product but a mixture of various
mono- and oligomers.
[0024] An alternative process for preparing glucosamine with the
aid of recombinant microorganisms, in particular Escherichia coli,
which does not require the degradation of chitin, is described in
US 2002/0160459.
[0025] For some time, glucosamine and glucosamine-containing
substances, too, have been considered as potential active compounds
for the treatment of osteoarthritis (Clegg et al., 2006, The New
England Journal of Medicine 354(8), 795-808). Glucosamine or
glucosamine-containing substances are also present in many food
supplements. Foods enriched with GlcNAc are described, for example,
in US 2006/0003965.
[0026] As already described, glucosaminoglycans, such as, for
example chondroitin sulfate, heparin/heparan sulfate or chitin are
currently isolated from animal tissues. In addition to the
substances desired in each case, these tissues also contain other
glucosaminoglycans. The separation of the individual
glucosaminoglycans, if a complete separation is possible at all, is
difficult and complicated. Furthermore, the potential presence, in
animal tissues, of pathogenic microorganisms and/or of other
substances, such as, for example, the BSE pathogen or the bird flu
pathogen, which may cause diseases in man, represent a problem when
using glucosaminoglycans isolated from animal tissue. The use of
medicinal preparations contaminated with animal proteins may, in
the patient, result in unwanted immunological reactions of the body
(for hyaluronan preparations, see, for example, U.S. Pat. No.
4,141,973), in particular if the patient is allergic to animal
proteins.
[0027] A further problem during the isolation of glucosaminoglycans
from animal tissues consists in the fact that the molecular weight
of the glucosaminoglycans is frequently reduced during
purification, since animal tissues also contain enzymes which
degrade glucosaminoglycan.
[0028] Glucosamine or derivatives thereof isolated from crustaceans
frequently contain substances (proteins) which may trigger an
allergic reaction in man. Glucosamine or derivatives obtained from
fungi may contain mycotoxins.
[0029] The amounts (yields) of glucosaminoglycans which can be
obtained in satisfactory quality and purity from animal tissues are
low (for example hyaluronan from roostercombs: 0.079% w/w, EP
0144019, U.S. Pat. No. 4,782,046), which means that large amounts
of animal tissues have to be processed.
[0030] The production of glucosaminoglycans with the aid of
fermentation of bacteria is associated with high costs, since the
bacteria have to be fermented in sealed sterile containers under
complicated controlled cultivation conditions (for hyaluronan, see,
for example, U.S. Pat. No. 4,897,349). Furthermore, the amount of
glucosaminoglycans which can be produced by fermentation of
bacteria strains is limited by the existing production facilities.
Here, it has also been taken into account that, owing to physical
limitations, it is not possible to construct fermenters for
relatively large culture volumes. In this context, mention may be
made in particular of homogeneous mixing, required for efficient
production, of fed-in substances (for example essential nutrient
sources for bacteria, reagents for regulating the pH, oxygen) with
the culture medium, which, if at all, can be ensured in large
fermenters only with high technical expenditure.
[0031] Furthermore, substances prepared from animal raw materials
are unacceptable for certain ways of life, such as, for example,
veganism or for kosher food preparation.
[0032] Plants do not naturally produce glucosaminoglycans, such as,
for example, hyaluronan, chitin, heparan/heparin sulfate, keratan
sulfate or chondroitin/dermatan sulfate.
[0033] For the synthesis of glucosaminoglycans, it is necessary,
inter alia, for sufficient amounts of acetylated glucosamine
derivatives (in particular UDP-GlcNAc) and/or UDP-GlcA to be
available as substrate for the respective enzymes involved in the
synthesis. There is no information with regard to the amounts of
N-acetylated glucosamines present in plant cells. WO 2005 035710
describes a process which allows the glucosamine content of plant
material to be increased by drying. The highest glucosamine content
in fresh, wet plant material was determined for chicory with 10 mg
of glucosamine per 1 kg of fresh weight, which, at a molecular
weight of 178 for glucosamine, corresponds to about 56 nmol of
glucosamine per 1 gram fresh weight of plant material. WO 2005
035710 contains no information concerning the content of
N-acetylated glucosamine derivatives in plants.
[0034] Furthermore, from the prior art described above, it is
evident that the paths of glucosamine metabolism in plants have not
yet been fully elucidated. In WO 00 11192, it was possible to
generate plants by transformation with a nucleic acid molecule
coding for a protein having the activity of a plant GFAT, which
plants had an elevated content of glucosamine derivatives
(UDP-glucosamine or glucosamine 1-phosphate); however, increased
amounts of N-acetylated glucosamine derivatives were not found.
[0035] Accordingly, it is an object of the present invention to
provide alternative sources of N-acetylated glucosamine derivatives
and processes for preparing said alternative sources for
N-acetylated glucosamine derivatives.
[0036] A first aspect of the present invention relates to plant
cells or plants having a content of N-acetylated glucosamine
derivatives of at least 2.50 .mu.mol per gram of fresh weight,
preferably of at least 5.00 .mu.mol per gram of fresh weight,
particularly preferably of at least 10.00 .mu.mol per gram of fresh
weight, very particularly preferably of at least 15.00 .mu.mol per
gram of fresh weight, especially preferably of at least 20.00
.mu.mol per gram of fresh weight.
[0037] Preferably, plant cells according to the invention or plants
according to the invention have a content of N-acetylated
glucosamine derivatives of at most 250 .mu.mol per gram of fresh
weight, preferably of at most 200 .mu.mol per gram of fresh weight,
particularly preferably of at most 150 .mu.mol per gram of fresh
weight, very particularly preferably of at most 100 .mu.mol per
gram of fresh weight, especially preferably of at most 50 .mu.mol
per gram of fresh weight.
[0038] Compared to the prior art, plant cells according to the
invention or plants according to the invention offer the advantage
that they contain higher amounts of N-acetylated glucosamine
derivatives. Compared to the production of N-acetylated glucosamine
derivatives by fermentation of microorganisms or the isolation of
N-acetylated glucosamines from animal raw material sources or
fungi, plant cells according to the invention or plants according
to the invention of the present invention offer the advantage that
plant cells according to the invention and plants according to the
invention can be propagated infinitely in a vegetative or sexual
manner, and that they continuously produce N-acetylated glucosamine
derivatives. Furthermore, compared to known plants, plants
according to the invention offer the advantage that they are better
suitable for preparing glucosaminoglycans, such as, for example,
chondroitin, hyaluronan, chitin, heparosan, since they contain a
higher amount of substrates for the enzymes involved in the
catalysis of the glucosaminoglycans mentioned (glucosaminoglycan
synthases).
[0039] N-Acetylated glucosamine derivatives can be detected using
methods known to the person skilled in the art (Morgan and Elson
(1934, Biochem J. 28(3), 988-995). In the context of the present
invention, for determining the content of N-acetylated glucosamine
derivatives, use is preferably made of the method described under
General Methods Item 4.
[0040] In the context of the present invention, the term
"N-acetylated glucosamine derivatives" is to be understood as
meaning all derivatives of glucosamine (2-amino-2-deoxyglucose),
which also include epimers, such as, for example, galactosamine
(2-amino-2-deoxygalactose) or mannosamine (2-amino-2-deoxymannose),
which are measured using the method described under General Methods
Item 4. The N-acetylated glucosamine derivatives are preferably
N-acetylglucosamine phosphate (N-acetylglucosamine 1-phosphate
and/or N-acetylglucosamine 6-phosphate), N-acetylglucosamine and/or
UDP-N-acetylglucosamine.
[0041] Preferrably plant cells according to the invention or plants
according to the invention have an increased content of glucosamine
phosphate (glucosamine 1-phosphate and/or glucosamine 6-phosphate)
in addition to an increased content of N-acetylated glucosamine
derivatives.
[0042] Plant cells according to the invention or plants according
to the invention can be prepared, for example, by introducing
foreign nucleic acid molecules coding for a protein having the
activity of a glutamine:fructose 6-phosphate amidotransferase
(GFAT) of isoform II (GFAT-2) or coding for a protein having the
activity of a bacterial GFAT.
[0043] In a preferred embodiment of the present invention, the
plant cells according to the invention or the plants according to
the invention are thus genetically modified plant cells and
genetically modified plants, respectively.
[0044] Surprisingly, it has been found that plant cells or plants
containing a nucleic acid molecule coding for a protein having the
activity of a GFAT-2 or a protein having the activity of a
bacterial GFAT contain considerably more N-acetylated glucosamine
derivatives than plant cells or plants containing a nucleic acid
molecule coding for a protein having the activity of a
glutamine:fructose 6-phosphate amidotransferase of isoform I
(GFAT-1). As already mentioned, it was not possible to detect
increased amounts of acetylated glucosamine derivatives in plants
containing a nucleic acid molecule coding for a protein having the
activity of a plant GFAT (WO 00 11192).
[0045] Accordingly, the present invention also provides genetically
modified plant cells or genetically modified plants containing a
foreign nucleic acid molecule coding for a protein having the
activity of a glutamine:fructose 6-phosphate amidotransferase
(GFAT), wherein the foreign nucleic acid molecule codes for a
protein having the activity of a glutamine:fructose 6-phosphate
amidotransferase of isoform II (GFAT-2) or a protein having the
activity of a bacterial glutamine:fructose 6-phosphate
amidotransferase (bacterial GFAT).
[0046] The genetic modification of a plant cell according to the
invention or a plant according to the invention may be any genetic
modification suitable for integrating a foreign nucleic acid
molecule into a plant cell or plant.
[0047] Preferably, the foreign nucleic acid molecule is integrated
into the genome; particularly preferably, the foreign nucleic acid
molecule is stably integrated into the genome of plant cells
according to the invention or plants according to the
invention.
[0048] A large number of techniques for (stably) integrating
nucleic acid molecules into a plant host cell for producing plant
cells according to the invention or plants according to the
invention is available. These techniques include the transformation
of plant cells with T-DNA using Agrobacterium tumefaciens or
Agrobacterium rhizogenes as means of transformation, protoplast
fusion, injection, electroporation of DNA, introduction of DNA by
the biolistic approach and also further options (review in
"Transgenic Plants", Leandro ed., Humana Press 2004, ISBN
1-59259-827-7).
[0049] The use of agrobacterium-mediated transformation of plant
cells has been subject to in-depth studies and has been described
exhaustively in EP 120516 and Hoekema, IN: The Binary Plant Vector
System Offsetdrukkerij Kanters B. V. Alblasserdam (1985), Chapter
V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and in An et al.
EMBO J. 4, (1985), 277-287. For the transformation of potatoes see,
for example, Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for the
transformation of tomato plants see, for example, U.S. Pat. No.
5,565,347.
[0050] The transformation of monocotyledonous plants using vectors
based on Agrobacterium transformation has been described, too (Chan
et al., Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J.
6, (1994) 271-282; Deng et al, Science in China 33, (1990), 28-34;
Wilmink et al., Plant Cell Reports 11, (1992), 76-80; May et al.,
Bio/Technology 13, (1995), 486-492; Conner and Domisse, Int. J.
Plant Sci. 153 (1992), 550-555; Ritchie et al, Transgenic Res. 2,
(1993), 252-265). Alternative systems for transforming
monocotyledonous plants are the transformation using the biolistic
approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil
et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant
Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet.
79, (1990), 625-631), the protoplast transformation, the
electroporation of partially permeabilized cells or the
introduction of DNA using glass fibers. In particular the
transformation of corn has been described several times in the
literature (cf., for example, WO95/06128, EP0513849, EP0465875,
EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844;
Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al.,
Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet.
80, (1990), 721-726). The transformation of other grasses, such as,
for example, switchgrass (Panicum virgatum) has also been described
(Richards et al., 2001, Plant Cell Reporters 20, 48-54).
[0051] The successful transformation of other cereal species has
likewise already been described, for example for barley (Wan and
Lemaux, loc. cit.; Ritala et al., loc. cit.; Krens et al., Nature
296, (1982), 72-74) and for wheat (Nehra et al., Plant J. 5,
(1994), 285-297; Becker et al., 1994, Plant Journal 5, 299-307).
All of the above methods are suitable in the context of the present
invention.
[0052] Genetically modified plant cells and genetically modified
plants having a foreign nucleic acid molecule can be distinguished
from wild-type plant cells and wild-type plants, respectively, not
having said foreign nucleic acid molecule, inter alia by the fact
that they contain a foreign nucleic acid molecule which does not
naturally occur in wild-type plant cells and wild-type plants,
respectively. Such an integration of a foreign nucleic acid
molecule into a plant cell or plant can be detected using methods
known to the person skilled in the art, such as, for example,
Southern blot analysis or by PCR.
[0053] In the context of the present invention, the term "stably
integrated nucleic acid molecule" is to be understood as meaning
the integration of a nucleic acid molecule into the genome of the
plant. A stably integrated nucleic acid molecule is characterized
in that, during the replication of the corresponding integration
site, it is multiplied together with the nucleic acid sequences of
the host which border on the integration site, so that the
integration site in the replicated daughter DNA strand is
surrounded by the same nucleic acid sequences as on the read mother
strand which serves as a matrix for the replication.
[0054] The integration of a nucleic acid molecule into the genome
of a plant cell or a plant can be demonstrated by genetic methods
and/or methods of molecular biology. A stable integration of a
nucleic acid molecule into the genome of a plant cell or into the
genome of a plant is characterized in that in the progeny which has
inherited said nucleic acid molecule, the stably integrated nucleic
acid molecule is present in the same genomic environment as in the
parent generation. The presence of a stable integration of a
nucleic acid sequence in the genome of a plant cell or in the
genome of a plant can be demonstrated using methods known to the
person skilled in the art, inter alia with the aid of Southern blot
analysis or the RFLP analysis (Restriction Fragment Length
Polymorphism) (Nam et al., 1989, The Plant Cell 1, 699-705; Leister
and Dean, 1993, The Plant Journal 4 (4), 745-750), with methods
based on PCR, such as, for example, the analysis of differences in
length in the amplified fragment (Amplified Fragment Length
Polymorphism, AFLP) (Castiglioni et al., 1998, Genetics 149,
2039-2056; Meksem et al., 2001, Molecular Genetics and Genomics
265, 207-214; Meyer et al., 1998, Molecular and General Genetics
259, 150-160) or using amplified fragments cleaved using
restriction endonucleases (Cleaved Amplified Polymorphic Sequences,
CAPS) (Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410;
Jarvis et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et
al., 1996, The Plant Journal 9 (5), 745-753).
[0055] In the context of the present invention, the term "genome"
is to be understood as meaning the entire genetic material present
in a plant cell. It is known to the person skilled in the art that,
in addition to the nucleus, other compartments (for example
plastids, mitochondria) also contain genetic material.
[0056] A further preferred subject matter of the present invention
relates to genetically modified plant cells according to the
invention or genetically modified plants according to the invention
expressing a foreign nucleic acid molecule coding for a protein
having the activity of a glutamine:fructose 6-phosphate
amidotransferase of isoform II (GFAT-2) or coding for a protein
having the activity of a bacterial glutamine:fructose 6-phosphate
amidotransferase (bacterial GFAT).
[0057] In the context of the present invention, the term "to
express" or "expression" is to be understood as meaning the
presence of transcripts (mRNA) coded for by a foreign nucleic acid
molecule and/or the presence of proteins having the activity of a
GFAT-2 or a bacterial GFAT.
[0058] An expression can be demonstrated, for example, by detection
of specific transcripts (mRNA) of foreign nucleic acid molecules by
Northern blot analysis or RT-PCR.
[0059] Whether plant cells or plants contain proteins having the
activity of a GFAT-2 or proteins having the activity of a bacterial
GFAT can be determined, for example, by immunological methods, such
as Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent
Assay) or RIA (Radio Immune Assay). The person skilled in the art
is familiar with methods for preparing antibodies which react
specifically with a certain protein, i.e. which bind specifically
to a certain protein (see, for example, Lottspeich and Zorbas
(eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg,
Berlin, ISBN 3-8274-0041-4). Some companies (for example
Eurogentec, Belgium) offer the preparation of such antibodies as an
order service.
[0060] In a further preferred embodiment of the present invention,
plant cells according to the invention or plants according to the
invention have an activity of a protein having the activity of a
glutamine:fructose 6-phosphate amidotransferase of isoform II
(GFAT-2) or of coding for a protein having the activity of a
bacterial glutamine:fructose 6-phosphate amidotransferase
(bacterial GFAT).
[0061] The activity of proteins having the activity of a GFAT-2 or
proteins having the activity of a bacterial GFAT in extracts of
plant cells according to the invention or plants according to the
invention can be detected using methods known to the person skilled
in the art, such as, for example, described in Samac et al. (2004,
Applied Biochemistry and Biotechnology 113-116, Humana Press,
Editor Ashok Mulehandani, 1167-1182, ISSN 0273-2289). A preferred
method for determining the amount of activity of a protein having
the activity of a GFAT is given in General Methods, Item 8.
[0062] In the context of the present invention, the term
"glutamine:fructose 6-phosphate amidotransferase (GFAT)" (E.C.
2.6.1.16), in the expert literature also referred to as glucosamine
synthase, is to be understood as meaning a protein which
synthesizes, from the starting materials glutamine and fructose
6-phosphate (Fruc-6-P), glucosamine 6-phosphate (GlcN-6-P). This
catalysis proceeds according to the following reaction scheme:
glutamine+Fruc-6-P.fwdarw.GlcN-6-P+glutamate
[0063] In the context of the present invention, the term
"glutamine:fructose 6-phosphate amidotransferase (GFAT)" is used as
a generic term which includes all known isoforms.
[0064] A review article by Milewski (2002, Biochimica et Biophysica
Acta 1597, 173-193) describes structural features of proteins
having the activity of a GFAT. The amino acid sequence of all known
proteins having the activity of GFAT contains regions with
conserved amino acid sequences. The amino acid sequence of proteins
having the activity of a GFAT has an N-terminal glutamine binding
domain and a C-terminal fructose 6-phosphate binding domain which
are separated by a sequence of 40 to 90 non-conserved amino acids.
Both domains are active even if they are present on separate amino
acid molecules. Analyses of the crystal structure of a fragment
comprising the N-terminal glutamine binding domain of the protein
having the activity of a GFAT from Escherichia coli showed that the
active center of this domain is located at the N-terminus and the
amino acid Cys1 is involved in the hydrolysis of glutamine. The
amino acids Arg73 and Asp123 interact with carboxyl and amino
groups of the glutamine. This interaction is supported by the amino
acids Thr76 and His77. The formation of hydrogen bonds with the
amido group of the glutamine is attributed to the amino acids Gly99
and Trp74. The amino acids Asn98 and Gly99 stabilize the four-faced
pocket of the active center. The amino acids 25 to 29 and 73-80
form flexible loops which, after binding of the substrate
glutamine, contribute by a conformational change of the protein to
the reaction catalyzed by a protein having the activity of a GFAT.
Analysis of the crystal structure of the C-terminal fructose
6-phosphate binding domain of the protein having the activity of a
GFAT from Escherichia coli showed that this domain is constructed
of two topologically identical domains (amino acids 241 to 424 and
425 to 592) followed by a domain present at the C-terminal end as
an irregular loop (amino acids 593 to 608), but which has only one
active center. The amino acids Ser303, Ser347, Gln348, Ser349 and
Thr352 are involved in substrate binding, whereas the amino acids
Glu488, His504 and Lys603 are directly involved in the catalysis of
the reaction of the protein having the activity of a GFAT.
[0065] In particular in animal organisms, it was possible to
demonstrate two different isoforms of proteins having the activity
of a GFAT (referred to in the literature as GFAT-1 and GFAT-2,
respectively). Hu et al. (2004, J. Biol. Chem. 279(29),
29988-29993) describe differences of the respective isoforms of
proteins having the activity of a GFAT. In addition to differences
in the tissue-specific expression of the isoforms in question
having the activity of a GFAT-1 and a GFAT-2, it was possible to
show that both isoforms are regulated by phosphorylation by a
cAMP-dependent protein kinase. The activity of a protein having the
enzymatic activity of a GFAT-1 is inhibited by phosphorylation of a
conserved serine residue (serine 205 in the GFAT-1 from the mouse,
GenBank Acc No.: AF334736.1) of the amino acid sequence in
question, whereas the activity of a protein having the activity of
a GFAT-2 is increased by phosphorylation of a conserved serine
residue (serine 202 in the GFAT-2 from the mouse, GenBank Acc No.:
NM.sub.--013529) of the amino acid sequence in question. Both
proteins having the activity of a GFAT-1 and proteins having the
activity of a GFAT-2 are inhibited in a concentration-dependent
manner by UDP-GlcNAc; however, for a protein having the activity of
a GFAT-2, the inhibition by UDP-GlcNAc is lower (maximum reduction
of activity by UDP-GlcNAc about 15%) compared to a protein having
the activity of a GFAT-1 (maximum reduction of activity by
UDP-GlcNAc by about 51% or 80%, respectively). There are
indications that the inhibition of a protein having the activity of
a GFAT-1 in animal organisms is based on the fact that at elevated
UDP-GlcNAc concentrations there is an O-glucose-N-acetylglucosamine
glycosylation of the proteins in question. Whether a regulation of
the activity of proteins by O-glycosylation also takes part in
plant cells is not yet fully understood (Huber and Hardin, 2004,
Current Opinion in Plant Biotechnology 7, 318-322).
[0066] Proteins having the activity of a bacterial GFAT are
distinguished by the fact that they are not inhibited by UDP-GlcNAc
(Kornfeld, 1967, J. Biol. Chem. 242(13), 3135-3141). Proteins
having the activity of a GFAT-1, proteins having the activity of a
GFAT-2, and even proteins having the activity of a bacterial GFAT
are inhibited by the product glucosamine 6-phosphate formed in
their reaction (Broschat et al., 2002, J. Biol. Chem. 277(17),
14764-14770; Deng et al., 2005, Metabolic Engineering 7,
201-214).
[0067] In the context of the present invention, the term "protein
having the activity of a glutamine:fructose 6-phosphate
amidotransferase of isoform I (GFAT-1)" is to be understood as
meaning a protein which has the activity of a GFAT and whose
activity is inhibited by phosphorylation by a cAMP-dependent
protein kinase.
[0068] In the context of the present invention, the term "protein
having the activity of a glutamine:fructose 6-phosphate
amidotransferase of isoform II (GFAT-2)" is to be understood as
meaning a protein which has the activity of a GFAT and which is
activated by phosphorylation by a cAMP-dependent protein
kinase.
[0069] In the context of the present invention, the term "protein
having the activity of a bacterial glutamine:fructose 6-phosphate
amidotransferase (bacterial GFAT)" is to be understood as meaning a
protein which has the activity of a GFAT and whose activity is not
inhibited by UDP-GlcNAc. Alternatively, "proteins having the
activity of a bacterial GFAT" may also be referred to as "proteins
having the activity of a non-eukaryotic GFAT".
[0070] In the context of the present invention, the term "foreign
nucleic acid molecule" is to be understood as meaning such a
molecule which either does not naturally occur in corresponding
wild-type plant cells or which does not naturally occur in the
specific spatial arrangement in wild-type plant cells or which is
localized at a site in the genome of the wild-type plant cell where
it does not naturally occur.
[0071] Preferably, the foreign nucleic acid molecule is a
recombinant molecule which consists of various elements (nucleic
acid molecules) whose combination or specific spatial arrangement
does not naturally occur in plant cells.
[0072] In the context of the present invention, the term
"recombinant nucleic acid molecule" is to be understood as meaning
a nucleic acid molecule which has various nucleic acid molecules
which are not naturally present in a combination as present in a
recombinant nucleic acid molecule. Thus, recombinant nucleic acid
molecules may, in addition to foreign nucleic acid molecules coding
for a protein, have, for example, additional nucleic acid sequences
which are not naturally present in combination with said
protein-encoding nucleic acid molecules. Here, the additional
nucleic acid sequences mentioned, which are present in a
recombinant nucleic acid molecule in combination with a
protein-encoding nucleic acid molecule, may be any sequences. They
may, for example, represent genomic and/or plant nucleic acid
sequences.
[0073] The additional nucleic acid sequences mentioned are
preferably regulatory sequences (promoters, termination signals,
enhancer, introns), particularly preferably regulatory sequences
active in plant tissue, very particularly preferably
tissue-specific regulatory sequences active in plant tissue.
[0074] Methods for generating recombinant nucleic acid molecules
are known to the person skilled in the art and include genetic
engineering methods, such as, for example, linking of nucleic acid
molecules by ligation, genetic recombination or the novel synthesis
of nucleic acid molecules (see, for example, Sambrok et al.,
Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ISBN:
0879695773; Ausubel et al., Short Protocols in Molecular Biology,
John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).
[0075] The present invention preferably provides genetically
modified plant cells according to the invention or genetically
modified plants according to the invention wherein the foreign
nucleic acid molecules coding for a protein having the activity of
a GFAT-2 or coding for a protein having the activity of a bacterial
GFAT are linked to regulatory elements initiating the transcription
in plant cells (promoters). These may be homologous or heterologous
promoters. The promoters may be constitutive, tissue-specific or
development-specific promoters or promoters regulated by external
factors (for example after the application of chemical substances,
by action of abiotic factors, such as heat and/or cold, dryness,
disease, etc.).
[0076] In general, any promoters active in plant cells are suitable
for expressing a foreign nucleic acid molecule. Suitable promoters
are, for example, the promoter of 35S RNA of the cauliflower mosaic
virus or the ubiquitin promoter from corn or the Cestrum YLCV
Promoter (Yellow Leaf Curling Virus; WO 01 73087; Stavolone et al.,
2003, Plant Mol. Biol. 53, 703-713) for a constitutive expression,
the patatingen promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989),
23-29) for a tuber-specific expression in potatoes or a
fruit-specific promoter for tomato, such as, for example, the
polygalacturonase promoter from tomato (Montgomery et al., 1993,
Plant Cell 5, 1049-1062) or the E8 promoter from tomato (Metha et
al., 2002, Nature Biotechnol. 20(6), 613-618) or the ACC oxidase
promoter from peach (Moon and Callahan, 2004, J. Experimental
Botany 55 (402), 1519-1528) or a promoter which ensures expression
only in photosynthetically active tissues, for example the ST-LS1
promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987),
7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or for an
endosperm-specific expression the HMWG promoter from wheat, the USP
promoter, the phaseolin promoter, promoters of zein genes from corn
(Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al.,
Plant Mol. Biol. 15 (1990), 81-93), a glutelin promoter (Leisy et
al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4
(1993), 357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218),
a globulin promoter (Nakase et al., 1996, Gene 170(2), 223-226), a
prolamin promoter (Qu and Takaiwa, 2004, Plant Biotechnology
Journal 2(2), 113-125) or a shrunken-1 promoter (Werr et al., EMBO
J. 4 (1985), 1373-1380). However, it is also possible to use
promoters which are only active at a point in time determined by
external factors (see, for example, WO 9307279). Of particular
interest here may be promoters of heat-shock proteins which permit
a simple induction. It is furthermore possible to use seed-specific
promoters, such as, for example, the USP promoter from Vicia faba
which ensures a seed-specific expression in Vicia faba and other
plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679;
Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).
[0077] The use of promoters present in the genome of
algae-infecting viruses is also suitable for expressing nucleic
acid sequences in plants (Mitra et al., 1994, Biochem. Biophys Res
Commun 204(1), 187-194; Mitra and Higgins, 1994, Plant Mol Biol
26(1), 85-93, Van Etten et al., 2002, Arch Virol 147,
1479-1516).
[0078] In the context of the present invention, the term "tissue
specific" is to be understood as meaning the substantial limitation
of a manifestation (for example initiation of transcription) to a
certain tissue.
[0079] In the context of the present invention, the terms "tuber,
fruit or endosperm cell" are to be understood as meaning all cells
present in a tuber, a fruit and in an endosperm of a seed,
respectively.
[0080] In the context of the present invention, the term
"homologous promoter" is to be understood as meaning a promoter
which is naturally present in plant cells or plants used for the
preparation of genetically modified plant cells according to the
invention and genetically modified plants according to the
invention, respectively, (homologous with respect to the plant cell
or the plant) or as meaning a promoter which regulates the
regulation of the expression of a gene in the organism from which
the respective foreign nucleic acid molecule coding for a protein
was isolated (homologous with respect to the nucleic acid molecule
to be expressed).
[0081] In the context of the present invention, the term
"heterologous promoter" is to be understood as meaning a promoter
which is not naturally present in plant cells or plants used for
the preparation of genetically modified plant cells according to
the invention and in genetically modified plants according to the
invention, respectively, (heterologous with respect to the plant
cell or plant) or as meaning a promoter which is, in the organism
from which the respective foreign nucleic acid molecule coding for
a protein was isolated, not naturally present for regulating the
expression of said foreign nucleic acid molecule (heterologous with
respect to the nucleic acid molecule to be expressed).
[0082] Also present may be a termination sequence (polyadenylation
signal) which serves to add a poly-A tail to the transcript. The
poly-A tail is thought to act in stabilizing the transcripts. Such
elements are described in the literature (cf. Gielen et al., EMBO
J. 8 (1989), 23-29) and can be exchanged as desired.
[0083] It is also possible for intron sequences to be present
between the promoter and the coding region of the foreign nucleic
acid molecule. Such intron sequences may lead to stability of
expression and an increased expression in plants (Callis et al.,
1987, Genes Devel. 1, 1183-1200; Luehrsen, and Walbot, 1991, Mol.
Gen. Genet. 225, 81-93; Rethmeier et al., 1997; Plant Journal
12(4), 895-899; Rose and Beliakoff, 2000, Plant Physiol. 122 (2),
535-542; Vasil et al., 1989, Plant Physiol. 91, 1575-1579; XU et
al., 2003, Science in China Series C Vol. 46 No. 6, 561-569).
Suitable intron sequences are, for example, the first intron of the
sh1 gene from corn, the first intron of the poly-ubiquitin gene 1
from corn, the first intron of the EPSPS gene from rice or one of
the first two introns of the PAT1 gene from Arabidopsis.
[0084] According to the invention, the foreign nucleic acid
molecule coding for a protein having the enzymatic activity of a
GFAT-2 may originate from any eukaryotic organism; preferably, said
nucleic acid molecule originates from animals, particularly
preferably from mammals and very particularly preferably from the
mouse.
[0085] According to the invention, the foreign nucleic acid
molecule coding for a protein having the enzymatic activity of a
bacterial GFAT may originate from any non-eukaryotic organism or
from a virus genome; preferably, said nucleic acid molecule
originates from bacteria or viruses; particularly preferably, said
nucleic acid molecule originates from Escherichia coli. Since amino
acid sequences coding for viral proteins having the activity of a
GFAT have a considerably higher identity with amino acid sequences
coding for proteins having the activity of a bacterial GFAT and a
considerably lower identity with proteins having the activity of a
GFAT-1 or a GFAT-2, viral proteins having the activity of a GFAT
are classed with the bacterial proteins having the activity of a
GFAT (Landstein et al., 1998, Virology 250, 388-396).
[0086] With regard to viruses, the foreign nucleic acid molecule
coding for a protein having the enzymatic activity of a GFAT
preferably originates from an algae-infecting virus, with
preference a virus which infects algae of the genus Chlorella,
particularly preferably from a Paramecium bursaria Chlorella virus
and very particularly preferably from a Paramecium bursaria
Chlorella virus of an H1 strain.
[0087] Instead of a naturally occurring nucleic acid molecule
coding for a protein having the activity of a GFAT-2 or coding for
a protein having the activity of a bacterial GFAT, a nucleic acid
molecule introduced into genetically modified plant cells according
to the invention or genetically modified plants according to the
invention may also have been generated by mutagenesis, where said
mutagenized foreign nucleic acid molecule is characterized in that
it codes for a protein having the activity of a GFAT-2 or a protein
having the activity of a bacterial GFAT which has reduced
inhibition by metabolites (for example of the glucosamine
metabolism). In an exemplary manner, the preparation of such
mutagenized nucleic acid molecules is described in Deng et al.
(2005, Metabolic Engineering 7, 201-214; WO 04 003175) for a
protein having the activity of a bacterial GFAT from Escherichia
coli. Mutants of a protein having the activity of a GFAT-2 from the
mouse are described, for example, in Hu et al. (2004, J. Biol.
Chem. 279 (29), 29988-29993).
[0088] Nucleic acid molecules coding for a protein having the
activity of a GFAT are known to the person skilled in the art and
described in the literature. Thus, nucleic acid molecules coding
for a protein having the activity of a bacterial GFAT are
described, for example, for Escherichia coli (Dutka-Malen, 1988,
Biochemie 70 (2), 287-290; EMBL acc No: L10328.1), Bacillus
subtilis (EMBL acc No U21932), Haemophilus influenzae (EMBL acc Nos
AB006424.1, BAA33071). Nucleic acid molecules coding for a protein
having the activity of a bacterial GFAT are also described for
viruses, such as, for example, the Chlorella virus k2 (EMBL acc No
AB107976.1).
[0089] Nucleic acid molecules coding for a protein having the
activity of a GFAT-2 are described inter alia from insects, for
example for Drosophila melanogaster (NCBI acc No
NM.sub.--143360.2), from vertebrates, for example for Homo sapiens
(NCBI acc No BC000012.2, Oki et al., 1999, Genomics 57 (2), 227-34)
or Mus musculus (EMBL acc No AB016780.1).
[0090] In a preferred embodiment, the present invention relates to
genetically modified plant cells according to the invention and
genetically modified plants according to the invention where the
foreign nucleic acid molecule coding for a protein having the
activity of a GFAT-2 or coding for a protein having the activity of
a bacterial GFAT is selected from the group consisting of [0091] a)
nucleic acid molecules coding for a protein having the amino acid
sequence given under SEQ ID NO 7 (GFAT-2) or a protein having the
amino acid sequence given under SEQ ID NO 9 (bacterial GFAT);
[0092] b) nucleic acid molecules coding for a protein whose
sequence is at least 60%, preferably at least 70%, more preferably
at least 80%, particularly preferably at least 90%, very
particularly preferably at least 95% and most preferably at least
98% identical to the amino acid sequence shown under SEQ ID NO 7
(GFAT-2) or under SEQ ID NO 9 (bacterial GFAT); [0093] c) nucleic
acid molecules comprising the nucleotide sequence shown under SEQ
ID NO 6 (GFAT-2) or under SEQ ID NO 8 (bacterial GFAT) or under SEQ
ID NO 10 (bacterial GFAT) or a sequence complementary thereto;
[0094] d) nucleic acid molecules which are at least 60%, preferably
at least 70%, more preferably at least 80%, particularly preferably
at least 90%, very particularly preferably at least 95% and most
preferably at least 98% identical to the nucleic acid sequences
shown under a) or c); [0095] e) nucleic acid molecules which
hybridize under stringent conditions with at least one strand of
the nucleic acid sequences described under a) or c); [0096] f)
nucleic acid molecules whose nucleotide sequence differs from the
sequence of the nucleic acid molecules mentioned under a) or c)
owing to the degeneracy of the genetic code and [0097] g) nucleic
acid molecules which are fragments, allelic variants and/or
derivatives of the nucleic acid molecules mentioned under a), b),
c), d), e) or f).
[0098] In the context of the present invention, the term
"hybridization" means a hybridization under conventional
hybridization conditions, preferably under stringent conditions, as
described, for example, in Sambrook et al. (Molecular Cloning, A
Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. ISBN: 0879695773) or Ausubel et al.
(Short Protocols in Molecular Biology, John Wiley & Sons; 5th
edition (2002), ISBN: 0471250929). With particular preference,
"hybridization" means a hybridization under the following
conditions:
hybridization buffer: 2.times.SSC; 10.times.Denhardt solution
(Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM
Na.sub.2HPO.sub.4; 250 .mu.g/ml of herring sperm DNA; 50 .mu.g/ml
of tRNA; or 25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS
hybridization temperature:
T=65 to 68.degree. C.
[0099] wash buffer: 0.1.times.SSC; 0.1% SDS wash temperature: T=65
to 68.degree. C.
[0100] Nucleic acid molecules which hybridize with nucleic acid
molecules coding for a protein having the activity of a GFAT-2 or
coding for a protein having the activity of a bacterial GFAT may
originate from any organism; accordingly, they may originate from
bacteria, fungi, animals, plants or viruses.
[0101] Nucleic acid molecules which hybridize with nucleic acid
molecules coding for a protein having the activity of a GFAT-2
preferably originate from animals, particularly preferably from
mammals and very particularly preferably from the mouse.
[0102] Nucleic acid molecules which hybridize with nucleic acid
molecules coding for a protein having the activity of a bacterial
GFAT preferably originate from bacteria or viruses, particularly
preferably from Escherichia coli.
[0103] Nucleic acid molecules which hybridize with the molecules
mentioned may be isolated, for example, from genomic or from cDNA
libraries. Such nucleic acid molecules can be identified and
isolated using the nucleic acid molecules mentioned or parts of
these molecules or the reverse complements of these molecules, for
example by hybridization according to standard methods (see, for
example, Sambrook et al., Molecular Cloning, A Laboratory Manual,
3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. ISBN: 0879695773; Ausubel et al., Short Protocols in
Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN:
0471250929) or by amplification using PCR.
[0104] As hybridization sample for isolating a nucleic acid
sequence coding for a protein having the activity of a GFAT-2, it
is possible to use, for example, nucleic acid molecules having
exactly or essentially the nucleic acid sequences described under
SEQ ID NO 6 or fragments of these nucleic acid sequences. As
hybridization sample for isolating a nucleic acid sequence coding
for a protein having the activity of a bacterial GFAT, it is
possible to use, for example, nucleic acid molecules having exactly
or essentially the nucleic acid sequences described under SEQ ID NO
8 or fragments of these nucleic acid sequences.
[0105] The fragments used as hybridization samples may also be
synthetic fragments or oligonucleotides prepared using the
customary synthesis techniques, whose sequence is essentially
identical to the nucleic acid molecule described in the context of
the present invention. Once genes which hybridize with the nucleic
acid sequences described in the context of the present invention
are identified and isolated, the sequence should be determined and
the properties of the proteins coded for by this sequence should be
analyzed to determine whether they are proteins having the activity
of a GFAT-2 or the activity of a bacterial GFAT. Methods of how to
determine whether a protein has the activity of a protein having
the activity of a GFAT-2 or having the activity of a bacterial GFAT
are known to the person skilled in the art and described, inter
alia, in the literature (bacterial GFAT: for example Deng et al.,
2005, Metabolic Engineering 7, 201-214; Kornfeld, 1967, J. Biol.
Chem. 242(13), 3135-3141; GFAT-2: for example Hu et al., 2004, J.
Biol. Chem. 279 (29), 29988-29993). The molecules hybridizing with
the nucleic acid molecules described in the context of the present
invention comprise in particular fragments, derivatives and allelic
variants of the nucleic acid molecules mentioned. In the context of
the present invention, the term "derivative" means that the
sequences of these molecules differ in one or more positions from
the sequences of the nucleic acid molecules described above and are
highly identical to these sequences. The differences to the nucleic
acid molecules described above may, for example, be due to deletion
(in particular deletion of N- and/or C-terminal regions), addition,
substitution, insertion or recombination.
[0106] In the context of the present invention, the term "identity"
means a sequence identity over the entire length of the coding
region of a nucleic acid molecule or the entire length of an amino
acid sequence coding for a protein of at least 60%, in particular
an identity of at least 70%, preferably of at least 80%,
particularly preferably of at least 90%, very particularly
preferably of at least 95% and most preferably at least 98%. In the
context of the present invention, the term "identity" is to be
understood as meaning the number of identical amino
acids/nucleotides (identity) with other proteins/nucleic acids,
expressed in percent.
[0107] Preferably, the identity with respect to a protein having
the activity of a GFAT-2 is determined by comparisons with the
amino acid sequence given under SEQ ID NO 7 and the identity with
respect to a nucleic acid molecule coding for a protein having the
activity of a GFAT-2 as determined by comparisons of the nucleic
acid sequence given under SEQ ID NO 6 with other proteins/nucleic
acids with the aid of computer programs. Preferably, the identity
with respect to a protein having the activity of a bacterial GFAT
is determined by comparisons of the amino acid sequence given under
SEQ ID NO 9 and the identity with respect to a nucleic acid
molecule coding for a protein having the activity of a bacterial
GFAT is determined by comparisons of the nucleic acid sequence
given under SEQ ID NO 8 or SEQ ID NO 10 with other proteins/nucleic
acids with the aid of computer programs. If sequences to be
compared with one another are of different length, the identity is
to be determined by determining the identity in percent of the
number of amino acids/nucleotides which the shorter sequence shares
with the longer sequence. Preferably, the identity is determined
using the known and publicly available computer program ClustalW
(Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).
ClustalW is made publicly available by Julie Thompson
(Thompson@EMBL-Heidelberg.DE) and Toby Gibson
(Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory,
Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also
be downloaded from various Internet pages, inter alia from IGBMC
(Institut de Genetique et de Biologie Moleculaire et Cellulaire,
B.P. 163, 67404 Illkirch Cedex, France;
ftp://ftp-igbmc.u-strasbg.fr/pub/) and from EBI
(ftp://ftp.ebi.ac.uk/pub/software/) and all mirrored Internet pages
of the EBI (European Bioinformatics Institute, Wellcome Trust
Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
[0108] Preferably, use is made of the ClustalW computer program of
version 1.8 to determine the identity between proteins described in
the context of the present invention and other proteins. Here, the
parameters have to be set as follows: KTUPLE=1, TOPDIAG=5,
WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8,
MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.
[0109] Preferably, use is made of the ClustalW computer program of
version 1.8 to determine the identity for example between the
nucleotide sequence of the nucleic acid molecules described in the
context of the present invention and the nucleotide sequence of
other nucleic acid molecules. Here, the parameters have to be set
as follows: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB,
GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS:unweighted.
[0110] Identity furthermore means that there is a functional and/or
structural equivalence between the nucleic acid molecules in
question or the proteins encoded by them. The nucleic acid
molecules which are homologous to the molecules described above and
represent derivatives of these molecules are generally variations
of these molecules which represent modifications having the same
biological function, i.e. coding for a protein having the activity
of a GFAT-2 or the activity of a bacterial GFAT. They may be either
naturally occurring variations, for example sequences from other
species, or mutations, where these mutations may have occurred in a
natural manner or were introduced by systematic mutagenesis.
Furthermore, the variations may be synthetically produced
sequences. The allelic variants may be either naturally occurring
variants or synthetically produced variants or variants generated
by recombinant DNA techniques. A special form of derivatives are,
for example, nucleic acid molecules which differ from the nucleic
acid molecules described in the context of the present invention as
a result of the degeneracy of the genetic code.
[0111] In a further preferred embodiment, the present invention
relates to genetically modified plant cells according to the
invention or genetically modified plants according to the invention
where nucleic acid molecules coding for a protein having the
activity of a GFAT-2 or coding for a protein having the activity of
a bacterial GFAT are characterized in that the codons of said
nucleic acid molecules are different from the codons of the nucleic
acid molecules which code for said protein having the activity of a
GFAT-2 or said protein having the activity of a bacterial GFAT of
the parent organism. Particularly preferably, the codons of the
nucleic acid molecules coding for a protein having the activity of
a GFAT-2 or coding for a protein having the activity of a bacterial
GFAT are changed such that they are adapted to the frequency of use
of the codons of the plant cell or the plant into whose genome they
are integrated or to be integrated.
[0112] As a result of the degeneracy of the genetic code, amino
acids can be encoded by one or more codons. In different organisms,
the codons coding for an amino acid are used at different
frequencies. Adapting the codons of a coding nucleic acid sequence
to the frequency of their use in the plant cell or in the plant
into whose genome the sequence to be expressed is to be integrated
may contribute to an increased amount of translated protein and/or
to the stability of the mRNA in question in the particular plant
cells or plants. The frequency of use of codons in the plant cells
or plants in question can be determined by the person skilled in
the art by examining as many coding nucleic acid sequences of the
organism in question as possible for the frequency with which
certain codons are used for coding a certain amino acid. The
frequency of the use of codons of certain organisms is known to the
person skilled in the art and can be determined in a simple and
rapid manner using computer programs. Such computer programs are
publicly accessible and provided for free inter alia on the
Internet (for example http://gcua.schoedl.de/;
http://www.kazusa.or.jp/codon/;
http://www.entelechon.com/eng/cutanalysis.html). Adapting the
codons of a coding nucleic acid sequence to the frequency of their
use in the plant cell or in the plant into whose genome the
sequence to be expressed is to be integrated can be carried out by
in vitro mutagenesis or, preferably, by de novo synthesis of the
gene sequence. Methods for the de novo synthesis of nucleic acid
sequences are known to the person skilled in the art. A de novo
synthesis can be carried out, for example, by initially
synthesizing individual nucleic acid oligonucleotides, hybridizing
these with oligonucleotides complementary thereto, so that they
form a DNA double strand, and then ligating the individual
double-stranded oligonucleotides such that the desired nucleic acid
sequence is obtained. The de novo synthesis of nucleic acid
sequences including the adaptation of the frequency with which the
codons are used to a certain target organism can also be sourced
out to companies offering this service (for example Entelechon
GmbH, Regensburg, Germany).
[0113] All of the nucleic acid molecules mentioned are suitable for
producing plant cells according to the invention or plants
according to the invention.
[0114] The genetically modified plant cells according to the
invention or the genetically modified plants according to the
invention may, in principle, be plant cells and plants,
respectively, of any plant species, i.e. both monocotyledonous and
dicotyledonous plants. They are preferably crop plants, i.e. plants
cultivated by man for the purpose of feeding man and animal or for
producing biomass and/or for preparing substances for technical,
industrial purposes. The genetically modified plant cells according
to the invention or the genetically modified plants according to
the invention are particularly preferably corn, rice, wheat, rye,
oats, barley, manioc, potato, tomato, switchgrass (Panicum
virgatum), sago, mung beans, peas, sorghum, carrots, eggplant,
radish, oilseed rape, alfalfa, soybean, peanuts, cucumbers,
pumpkins, melons, leek, garlic, cabbage, spinach, sweet potato,
asparagus, zucchini, lettuce, artichokes, sweetcorn, parsnip,
salsify, Jerusalem artichoke, banana, sugar beet, sugar cane,
beetroot, broccoli, cabbage, onion, beet, dandelion, strawberry,
apple, apricot, plum, peach, grapevines, cauliflower, celery, bell
peppers, swede, rhubarb. They are preferably corn, rice, wheat,
rye, oat or barley plants, very particularly preferably rice,
tomato or potato plants.
[0115] In the context of the present invention, the term "potato
plant" or "potato" is to be understood as meaning plant species of
the genus Solanum, particularly tuber-producing species of the
genus Solanum and in particular Solanum tuberosum.
[0116] In the context of the present invention, the term "tomato
plant" or "tomato" is to be understood as meaning plant species of
the genus Lycopersicon, in particular Lycopersicon esculentum.
[0117] In the context of the present invention, the term "rice
plant" is to be understood as meaning plant species of the genus
Oryza, in particular plant species of the genus Oryza
agriculturally cultivated for commercial purposes, particularly
preferably Oryza saliva.
[0118] As already discussed, plant cells according to the invention
or plants according to the invention are suitable for producing
glucosaminoglycans, such as, for example, chondroitin, hyaluronan,
chitin, heparin (heparosan), since they contain a higher amount of
substrates for the enzymes involved in the catalysis of the
glucosaminoglycans mentioned.
[0119] Accordingly, the present invention furthermore relates to
plant cells or plants synthesizing glucosaminoglycan, preferably at
least 500 .mu.g of glucosaminoglycan per gram of fresh weight, more
preferably at least 1500 .mu.g of glucosaminoglycan per gram of
fresh weight, particularly preferably at least 3500 .mu.g of
glucosaminoglycan per gram of fresh weight, very particularly
preferably at least 4000 .mu.g of glucosaminoglycan per gram of
fresh weight and especially preferably at least 5500 .mu.g of
glucosaminoglycan per gram of fresh weight. In this context, the
glucosaminoglycan is preferably chondroitin, hyaluronan, chitin or
heparin (heparosan), particularly preferably hyaluronan.
[0120] Plant cells according to the invention or plants according
to the invention preferably have a glucosaminoglycan content of at
most 25 000 .mu.mol per gram of fresh weight, preferably at most 20
000 .mu.mol per gram of fresh weight, particularly preferably at
most 15 000 .mu.mol per gram of fresh weight, very particularly
preferably at most 10 000 .mu.mol per gram of fresh weight,
especially preferably at most 6500 .mu.mol per gram of fresh
weight.
[0121] Plant cells according to the invention or plants according
to the invention which synthesize glucosaminoglycan can be
produced, for example, by introducing foreign nucleic acid
molecules coding for a protein having the activity of a GFAT and
coding for a protein having the activity of a glucosaminoglycan
synthase into a plant cell.
[0122] Accordingly, the present invention also relates to
genetically modified plant cells or genetically modified plants
containing a first foreign nucleic acid molecule coding for a
protein having the activity of a GFAT-2 or a bacterial GFAT and a
second foreign nucleic acid molecule coding for a protein having
the activity of a glucosaminoglycan synthase.
[0123] In the context of the present invention, the term "protein
having the activity of a glucosaminoglycan synthase" is to be
understood as meaning a protein which uses UDP-GlcNAc or
UDP-N-acetylgalactosamine, an epimer of UDP-GlcNAc, as substrate
for synthesizing a glucosaminoglycan. The protein having the
activity of a glucosaminoglycan synthase is preferably a hyaluronan
synthase, chondroitin synthase, heparosan/heparin synthase, keratan
synthase or chitin synthase.
[0124] Nucleic acid molecules and corresponding protein sequences
coding for glucosaminoglycan synthases are known to the person
skilled in the art and described as hyaluronan synthase for example
from viruses (for example Paramecium bursaria Chlorella Virus 1,
EMBL U42580.3, PB42580, US 20030235893), as chondroitin synthase
for example from mammals (for example Homo sapiens, WO 03 012099,
US 2005048604, US 2006052335), bacteria (for example Escherichia
coli, US2003109693, EP 1283259, Pasteurella multicoda US
2003104601), as chitin synthase for example from bacteria (for
example Azorhizobium caulinodans EMBLCDS:AAB51164), from fungi (for
example Chaetomium globosum EMBLCDS:EAQ92361, Aspergillus nidulans
EMBL AB00125, Arthroderma benhamiae EMBLCDS:BAB32692 Neurospora
crassa EMBL M73437.4), from insects (for example Aedes aegypti
EMBLCDS:EAT46081, Tribolium castaneum EMBLCDS: AAQ55061), nematodes
(for example Dirofilaria immitis EMBL AF288618, Caenorhabditis
elegans EMBL AY874871), from viruses (for example Chlorella virus
EMBLCDS: BAB83509, Paramecium bursaria Chlorella virus CVK2
EMBLCDS: BAE48153), as heparin/heparosan synthase for example from
bacteria (for example Pasteurella multocida EMBL AF425591,
AF439804, US 20030099967, Escherichia coli X77617.1).
[0125] The second foreign nucleic acid molecule coding for a
protein having the activity of a glucosaminoglycan synthase is
preferably a recombinant nucleic acid molecule. Preferred
embodiments of recombinant nucleic acid molecules have already been
described and are to be used here in a corresponding manner.
[0126] In a further preferred embodiment, the second foreign
nucleic acid molecule coding for a protein having the activity of a
glucosaminoglycan synthase is characterized in that the codons are
modified compared to the codons of the nucleic acid molecule coding
for said protein having the activity of a glucosaminoglycan
synthase of the parent organism. Particularly preferably, the
codons of the nucleic acid molecules coding for a protein having
the activity of a glucosaminoglycan synthase are modified such that
they are adapted to the frequency of use of the codons of the plant
cell or the plant into whose genome they are integrated or to be
integrated.
[0127] What was stated above for nucleic acid molecules coding for
a protein having the activity of a GFAT-2 or a bacterial GFAT with
respect to the modification of the codons of a nucleic acid
molecule is to be applied here in a corresponding manner.
[0128] The present invention furthermore relates to plants
containing plant cells according to the invention. Such plants may
be generated by regeneration from plant cells according to the
invention.
[0129] The present invention also relates to parts of plants
according to the invention containing plant cells according to the
invention.
[0130] In the context of the present invention, the term "plant
parts" or "parts of plants" is to be understood as meaning, for
example, processible plant parts used in the production of
foodstuff or feedstuff, used as raw material source for industrial
processes (for example for the isolation of glucosamine derivatives
or glucosaminoglycans), as raw material source for the preparation
of pharmaceuticals or as raw material source for the preparation of
cosmetic products.
[0131] In the context of the present invention, the term "plant
parts" or "parts of plants" is furthermore to be understood as
meaning, for example, consumable plant parts which serve as food
for man or which are used as animal feed.
[0132] Preferred "plant parts" or "parts of plants" are fruits,
storage and other roots, flowers, buds, shoots, leaves or stalks,
preferably seeds, fruits, grains or tubers.
[0133] The present invention also relates to propagation material
of plants according to the invention. Preferably, propagation
material according to the invention contains plant cells according
to the invention, particularly preferably genetically modified
plant cells according to the invention.
[0134] Here, the term "propagation material" comprises those
components of the plant which are suitable for generating progeny
via the vegetative or generative route. Suitable for vegetative
propagation are, for example, cuttings, callus cultures, rhizomes
or tubers. Other propagation material includes, for example,
fruits, seeds, grains, seedlings, cell cultures, etc. The
propagation material preferably takes the form of tubers, fruits,
grains or seeds.
[0135] A further advantage of the present invention is the fact
that parts of plants according to the invention have a higher
content of N-acetylated glucosamine derivatives than known plants.
Accordingly, plants according to the invention are particularly
suitable for direct use as foodstuff/feedstuff or for preparing
foodstuff/feedstuff having a prophylactic or therapeutic effect
(for example for osteoarthritis prophylaxis). Since plants
according to the invention have a higher content of N-acetylated
glucosamine derivatives compared to known plants, the amounts of
harvestable parts, propagation material, processible parts or
consumable parts of plants according to the invention used for
preparing foodstuff/feedstuff having an increased content of
N-acetylated glucosamine derivatives can be reduced. If consumable
parts of genetically modified plants according to the invention are
consumed, for example, directly as so-called nutraceutical, a
positive effect may be achieved even by the consumption of small
amounts of substance. This may be of particular importance inter
alia in the production of animal feed since animal feed with too
high a content of plant components is unsuitable as feedstuff for
various animal species. Furthermore, plant cells according to the
invention or plants according to the invention have the advantage
that they can also be used by vegans or for preparing kosher food.
It is thus possible to administer food having an elevated content
of N-acetylated glucosamines even to people following the ways of
life mentioned.
[0136] It is known that N-acetylglucosamine has a stimulating
effect on the growth of bifido bacteria (Liepke et al., 2002, Eur.
J. Biochem. 269, 712-718). Furthermore, it has been shown that
N-acetylglucosamine serves as a substrate for lactobacilli (for
example Lactobacillus casei subspecies paracasei) from fish gut
(Adolfo Bucio Galindo, 2004, Proefschrift, Wageningen Universiteit,
ISBN 90-5808-943-6). Accordingly, N-acetylglucosamine has a
positive effect on probiotic bacteria. Since plant cells according
to the invention, plants according to the invention or parts of
plants according to the invention have an elevated content of
N-acetylglucosamine, they should consequently have a positive
effect on the growth of probiotic bacteria and thus be suitable for
use as a prebiotic foodstuff/feedstuff for man and animal.
[0137] The present invention furthermore relates to a process for
producing a genetically modified plant which comprises the
following steps: [0138] a) introduction of a foreign nucleic acid
molecule coding for a protein having the activity of a
glutamine:fructose 6-phosphate amidotransferase of isoform II
(GFAT-2) or coding for a protein having the activity of a bacterial
glutamine:fructose 6-phosphate amidotransferase (bacterial GFAT)
into a plant cell [0139] b) regeneration of a plant from plant
cells obtained according to step a) [0140] c) if appropriate,
generation of further plants with the aid of the plants according
to step b).
[0141] The present invention furthermore relates to processes for
producing a plant which synthesizes glucosaminoglycan, wherein
[0142] a) a plant cell is genetically modified, where the genetic
modification comprises the following steps i to ii in any order or
carrying out any combinations of the following steps i to ii
individually or simultaneously [0143] i) introducing a foreign
nucleic acid molecule coding for a protein having the activity of a
glutamine:fructose 6-phosphate amidotransferase of isoform II
(GFAT-2) or coding for a protein having the activity of a bacterial
glutamine:fructose 6-phosphate amidotransferase (bacterial GFAT)
into a plant cell [0144] ii) introducing a foreign nucleic acid
molecule coding for a glucosaminoglycan synthase into a plant cell
[0145] b) a plant is regenerated from plant cells comprising the
genetic modification according to steps [0146] i) a) i [0147] ii)
a) ii [0148] iii) a) i and a) ii, [0149] c) introducing into plant
cells of plants according to step [0150] i) b) i a genetic
modification according to step a) ii, [0151] ii) b) ii a genetic
modification according to step a) i, and regenerating a plant
[0152] d) if appropriate, generating further plants with the aid of
the plants obtained according to any of steps b) iii or c) i or c)
ii.
[0153] With regard to the introduction of foreign nucleic acid
molecules according to step a) of the process for producing a
genetically modified plant or according to steps a) or c) of the
process for producing a plant which synthesizes glucosaminoglycan
into a plant cell, this introduction may, in principle, be any type
of introduction of nucleic acid molecules suitable for integrating
a foreign nucleic acid molecule into a plant cell or plant. Such
methods have already been described above and can be applied here
in a corresponding manner.
[0154] With respect to the foreign nucleic acid molecule coding for
a protein having the activity of a GFAT-2 or coding for a protein
having the activity of a bacterial GFAT according to step a) of the
process for producing a genetically modified plant or with respect
to the foreign nucleic acid molecule coding for a protein having
the activity of a glucosaminoglycan synthase according to step a)
ii) of the process for producing a plant which synthesizes
glucosaminoglycan, various possible embodiments of the respective
nucleic acid molecules have already been described in the context
with plant cells according to the invention and plants according to
the invention. All these preferred embodiments which have already
been described can also be used for carrying out the processes
according to the invention mentioned.
[0155] The regeneration of the plants depending on the process
according to step b) and/or c) of the processes according to the
invention can be carried out using methods known to the person
skilled in the art (described, for example, in "Plant Cell Culture
Protocols", 1999, edt. by R. D. Hall, Humana Press, ISBN
0-89603-549-2).
[0156] The generation of further plants depending on the process
according to the step c) or d) of the processes according to the
invention can be carried out, for example, by vegetative
propagation (for example via cuttings, tubers or via callus culture
and regeneration of intact plants) or via generative propagation.
In this context, generative propagation preferably takes place
under controlled conditions, i.e. selected plants with specific
characteristics are hybridized with one another and multiplied. The
selection preferably takes place in such a manner that the plants,
depending on the process according to step b) or d), have the
modifications introduced in step a).
[0157] In a further preferred embodiment, processes according to
the invention for producing a genetically modified plant are used
for producing plants according to the invention.
[0158] The present invention also provides plants obtainable by
processes according to the invention for preparing a genetically
modified plant.
[0159] The present invention furthermore relates to a process for
producing glucosaminoglycans which comprises the step of the
extraction of glucosaminoglycans from genetically modified plant
cells according to the invention, from genetically modified plants
according to the invention, propagation material according to the
invention, parts of plants according to the invention or plants
obtainable by a process according to the invention for preparing a
genetically modified plant which synthesizes glucosaminoglycan. The
process according to the invention is preferably used for producing
chondroitin, hyaluronan, chitin or heparin (heparosan),
particularly preferably for producing hyaluronan.
[0160] Preferably, such a process also comprises the step of
harvesting the cultivated genetically modified plant cells
according to the invention, the genetically modified plants
according to the invention, the propagation material according to
the invention, the parts of plants according to the invention prior
to the extraction of the glucosaminoglycan and particularly
preferably furthermore the step of the cultivation of genetically
modified plant cells according to the invention or genetically
modified plants according to the invention prior to harvesting.
[0161] In contrast to bacterial or animal tissues, plant tissues do
not contain any glucosaminoglycan-degrading enzymes. Accordingly,
extraction of glucosaminoglycans from plant tissue is possible
using relatively simple methods. If required, aqueous extracts of
plant cells or tissues containing glucosaminoglycan can be purified
further using methods known to the person skilled in the art, such
as, for example, repeated precipitation with ethanol. A preferred
method for purifying, for example, hyaluronan is described under
General Methods Item 5.
[0162] The present invention also provides the use of genetically
modified plant cells according to the invention, genetically
modified plants according to the invention, propagation material
according to the invention, parts of plants according to the
invention or plants obtainable by a process according to the
invention for producing a genetically modified plant which
synthesizes glucosaminoglycan for producing glucosaminoglycans.
[0163] The present invention also provides the use of nucleic acid
molecules coding for a protein having the activity of a GFAT-2 or
coding for a protein having the activity of a bacterial GFAT for
preparing a genetically modified plant.
[0164] The present invention furthermore relates to a composition
comprising genetically modified plant cells according to the
invention.
[0165] Here, it is immaterial whether the plant cells are intact or
no longer intact because they have been destroyed, for example, by
processing. The compositions are preferably foodstuff, food
supplements or feedstuff, pharmaceutical or cosmetic products.
[0166] The present invention preferably provides compositions
according to the invention comprising recombinant nucleic acid
molecules, the recombinant nucleic acid molecules being
characterized in that they comprise nucleic acid molecules coding
for a protein having the enzymatic activity of a GFAT-2 or a
protein having the activity of a bacterial GFAT.
[0167] A stable integration of foreign nucleic acid molecules into
the genome of a plant cell or plant results in the foreign nucleic
acid molecules being flanked after integration into the genome of
the plant cell or plant by genomic plant nucleic acid sequences.
Accordingly, in a preferred embodiment, compositions according to
the invention are characterized in that the recombinant nucleic
acid molecules present in the composition according to the
invention are flanked by genomic plant nucleic acid sequences.
[0168] Here, the genomic plant nucleic acid sequences may be any
sequences naturally present in the genome of the plant cell or
plant used for preparing the composition. That recombinant nucleic
acid molecules which are present in the compositions according to
the invention can be demonstrated using methods known to the person
skilled in the art, such as, for example, methods based on
hybridization or, preferably, methods based on PCR (Polymerase
Chain Reaction).
[0169] Preferably, the compositions according to the invention
comprise at least 0.05%, preferably at least 0.1%, particularly
preferably at least 0.5%, very particularly preferably at least
1.0%, of N-acetylated glucosamine derivatives.
[0170] Preferably, the compositions according to the invention
comprise at most 10%, preferably at most 5%, particularly
preferably at most 3%, very particularly preferably at most 2%, of
N-acetylated glucosamine derivatives.
[0171] Compositions according to the invention offer the advantage
that they have an increased content of N-acetylated glucosamine
derivatives or an increased content of glucosaminoglycans compared
to compositions comprising not genetically modified plant cells.
N-Acetylglucosamine has a stimulating effect on the growth of
bifido bacteria (Liepke et al., 2002, Eur. J. Biochem. 269,
712-718). Furthermore, it has been shown that N-acetylglucosamine
serves as substrate for lactobacilli (for example Lactobacillus
casei subspecies paracasei) from fish gut (Adolfo Bucio Galindo,
2004, Proefschrift, Wageningen Universiteit, ISBN 90-5808-943-6).
Accordingly, N-acetylglucosamine has a positive effect on probiotic
bacteria. Since compositions according to the invention have
increased N-acetylglucosamine contents, they should have a positive
effect on the growth of probiotic bacteria.
[0172] The invention furthermore provides processes for preparing a
composition according to the invention using plant cells according
to the invention, plants according to the invention, propagation
material according to the invention, parts of plants according to
the invention or plants obtainable by a process according to the
invention for producing a genetically modified plant. The processes
for preparing a composition according to the invention are
preferably processes for producing foodstuff, feedstuff or food
supplements.
[0173] Processes for producing foodstuff, feedstuff, food
supplements, pharmaceutical products or cosmetic products are known
to the person skilled in the art and comprise inter alia, but are
not exclusively limited to, the comminuting or the grinding of
plants according to the invention or plant parts according to the
invention.
[0174] The present invention also provides compositions obtainable
by a process for preparing a composition according to the
invention.
[0175] The present invention also relates to the use of genetically
modified plant cells according to the invention or genetically
modified plants according to the invention for preparing a
composition according to the invention.
[0176] A preferred embodiment of compositions according to the
invention are flours.
[0177] Parts of plants are frequently processed to flours. Examples
of parts of plants which are used to prepare flours are, for
example, tubers of potato plants and grains of cereal plants. To
produce flours from cereal plants, the endosperm-containing grains
of these plants are ground and sieved. In the case of other plants
which do not contain any endosperm but, for example, tubers or
storage roots, flour is often produced by comminuting, drying and
subsequent grinding of the relevant parts of the plants. Plant
cells according to the invention and plants according to the
invention have an increased content of N-acetylated glucosamine
derivatives or glucosaminoglycans compared to known plant cells or
plants. Flours prepared from plant cells according to the
invention, plants according to the invention, propagation material
according to the invention or parts of plants according to the
invention accordingly likewise contain an increased proportion of
N-acetylated glucosamine derivatives or glucosaminoglycans.
[0178] Accordingly, the present invention furthermore relates to
flours which obtainable from plant cells according to the
invention, plants according to the invention or from parts of
plants according to the invention. Preferred parts of plants
according to the invention for producing flours are tubers and
endosperm-containing grains. In the context of the present
invention, particular preference is given to grains of plants of
the (systematic) family Poaceae, especially preferably, the grains
originate from corn, rice or wheat plants.
[0179] The present invention furthermore relates to flours
according to the invention having a content of N-acetylated
glucosamine derivatives of at least 10 .mu.mol per gram, preferably
at least 20 .mu.mol per gram, more preferably at least 25 .mu.mol
per gram, particularly preferably at least 30 .mu.mol per gram,
very particularly preferably at least 35 .mu.mol per gram and
especially preferably at least 40 .mu.mol per gram.
[0180] Flours according to the invention preferably have a content
of N-acetylated glucosamine derivatives of at most 250 .mu.mol per
gram of fresh weight, preferably at most 200 .mu.mol per gram of
fresh weight, particularly preferably at most 150 .mu.mol per gram
of fresh weight, very particularly preferably at most 100 .mu.mol
per gram of fresh weight and especially preferably at most 50
.mu.mol per gram of fresh weight.
[0181] In the context of the present invention, the term "flour" is
to be understood as meaning a powder obtained by grinding plants or
plant parts. If appropriate, the plants or plant parts are dried
prior to grinding and, after grinding, further comminuted and/or
sieved.
[0182] Compared to conventional flours, flours according to the
invention have the advantage that they can be used for producing
foodstuff, such as, for example, baked goods, having an increased
content of N-acetylated glucosamine derivatives or
glucosaminoglycans without it being necessary to add N-acetylated
glucosamine derivatives or glucosaminoglycans obtained from animal
or fungal raw material sources to the flour. The disadvantages of
the use of N-acetylated glucosamine derivatives or
glucosaminoglycans isolated from the raw material sources
mentioned, such as, for example, the risk that they may contain
pathogens or allergenic substances, have already been mentioned
further above.
[0183] The present invention furthermore provides a process for
producing flours which comprises the step of grinding plant cells
according to the invention, plants according to the invention or
parts of plants according to the invention.
[0184] Flours can be produced by grinding parts of plants. It is
known to the person skilled in the art how to produce flours. A
process for producing flours preferably also comprises the step of
harvesting the cultivated plants according to the invention or
parts of plants according to the invention and/or the propagation
material according to the invention and particularly preferably
furthermore the step of the cultivation of plants according to the
invention prior to harvesting.
[0185] In a further embodiment of the present invention, the
process for producing flours comprises processing of plants
according to the invention, of parts of plants according to the
invention or of propagation material according to the
invention.
[0186] Here, processing may, for example, be heat treatment and/or
drying. Heat treatment followed by drying of the heat-treated
material is used, for example, when producing flours from storage
roots, tubers such as, for example, from potato tubers, prior to
grinding. Comminuting of plants according to the invention, of
parts of plants according to the invention or of propagation
material according to the invention may also constitute processing
in the sense of the present invention. Removal of plant tissue,
such as, for example, removal of the husk from the grains, prior to
grinding also constitutes processing prior to grinding in the sense
of the present invention.
[0187] In a further embodiment of the present invention, the
process for producing flours comprises processing of the ground
material after grinding.
[0188] Here, the ground material may, for example, be sieved after
grinding, for example to produce different types of flour.
[0189] The present invention furthermore provides the use of plant
cells according to the invention, plants according to the
invention, of parts of plants according to the invention or of
propagation material according to the invention for producing
flours.
Description of the Sequences
[0190] SEQ ID NO 1: Nucleic acid sequence coding for a hyaluronan
synthase of Paramecium bursaria Chlorella Virus 1. [0191] SEQ ID NO
2: Amino acid sequence of a hyaluronan synthase of Paramecium
bursaria Chlorella Virus 1. The amino acid sequence shown can be
derived from SEQ ID NO 1. [0192] SEQ ID NO 3: Synthetic nucleic
acid sequence coding for a hyaluronan synthase of Paramecium
bursaria Chlorella Virus 1. The codons of the sequence shown were
synthesized in a manner such that they are adapted to the use of
codons in plant cells. The nucleic acid sequence shown codes for a
protein having the amino acid sequence shown under SEQ ID NO 2.
[0193] SEQ ID NO 4: Nucleic acid sequence coding for a protein
having the activity of a GFAT-1 from the mouse. [0194] SEQ ID NO 5:
Amino acid sequence of a protein having the activity of a GFAT-1
from the mouse. The amino acid sequence shown can be derived from
SEQ ID NO 4. [0195] SEQ ID NO 6: Nucleic acid sequence coding for a
protein having the activity of a GFAT-2 from the mouse. [0196] SEQ
ID NO 7: Amino acid sequence of a protein having the activity of a
GFAT-2 from the mouse. The amino acid sequence shown can be derived
from SEQ ID NO 6. [0197] SEQ ID NO 8: Nucleic acid sequence coding
for a protein having the activity of a bacterial GFAT from
Escherichia coli. [0198] SEQ ID NO 9: Amino acid sequence of a
protein having the activity of a GFAT from Escherichia coli. The
amino acid sequence shown can be derived from SEQ ID NO 8. [0199]
SEQ ID NO 10: Synthetic nucleic acid sequence coding for a protein
having the activity of a GFAT from Escherichia coli. The codons of
the sequence shown were synthesized in a manner such that they are
adapted to the use of codons in plant cells. The nucleic acid
sequence shown codes for a protein having the amino acid sequence
shown under SEQ ID NO 9. [0200] SEQ ID NO 11: Nucleic acid sequence
coding for a protein having the activity of a UDP-glucose
dehydrogenase of Paramecium bursaria Chlorella Virus 1. [0201] SEQ
ID NO 12: Amino acid sequence of a protein having the activity of a
UDP-glucose dehydrogenase of Paramecium bursaria Chlorella Virus 1.
The amino acid sequence shown can be derived from SEQ ID NO 11.
[0202] SEQ ID NO 13: Synthetic nucleic acid sequence coding for a
protein having the activity of a UDP-glucose dehydrogenase of
Paramecium bursaria Chlorella Virus 1. The codons of the sequence
shown were synthesized in a manner such that they are adapted to
the use of codons in plant cells. The nucleic acid sequence shown
codes for a protein having the amino acid sequence shown under SEQ
ID NO 12. [0203] SEQ ID NO 14: Synthetic oligonucleotide which was
used in example 6. [0204] SEQ ID NO 15: Synthetic oligonucleotide
which was used in example 6. [0205] SEQ ID NO 16: Synthetic
oligonucleotide which was used in example 15. [0206] SEQ ID NO 17:
Synthetic oligonucleotide which was used in example 15.
[0207] The content of all cited publications including the
accession numbers of nucleic acid molecules and amino acid
sequences mentioned for sequence databases is incorporated by
reference into the description of the application.
[0208] Methods which can be used in connection with the present
invention are described below. These methods are specific
embodiments; however, the present invention is not limited to these
methods. It is known to the person skilled in the art that the
invention can be carried out in the same manner by modifying the
methods described and/or by replacing individual methods or parts
of methods by alternative methods or alternative parts of
methods.
General Methods
1. Transformation of Potato Plants
[0209] Potato plants were transformed with the aid of
Agrobacterium, as described in Rocha-Sosa et al. (EMBO J. 8,
(1989), 23-29).
2. Transformation of Tomato Plants
[0210] Tomato plants were transformed with the aid of Agrobacterium
according to the method described in U.S. Pat. No. 5,565,347.
3. Transformation of Rice Plants
[0211] Rice plants were transformed according to the method
described by Hiei et al. (1994, Plant Journal 6(2), 271-282).
4. Determination of the Content of N-Acetylated Glucosamines
[0212] N-Acetylated glucosamine derivatives having a reducing end
were determined similarly to the method of Elson and Morgan (1933,
J. Biochem. 27, 1824) and the improved calorimetric determination
method of Reissig et al. (1955, Biol. Chem. 217, 959-966). The
calorimetric determination method is based on a reaction of
chromogen III (Muckenschnabel et al., 1998, Cancer Letters 131,
13-20) with p-dimethylaminobenzaldehyde (DMAB, Ehrlich's reagent),
yielding a red product whose concentration can be determined
photometrically.
a) Work-Up of the Plant Material
[0213] First, harvested plant material was comminuted. Depending on
the amount of plant material used, comminution was carried out in a
laboratory oscillating ball mill (MM200, from Retsch, Germany) for
30 seconds at 30 Hz or using a Warring blender at maximum speed for
about 30 seconds. In general, 0.5 g of the comminuted plant
material (for example leaf, tuber or rice grain) was mixed with 1
ml of a solution consisting of 7% perchloric acid, 5 mM EGTA and
incubated on ice for 20 minutes. The mixture was then centrifuged
(5 minutes at 16 000.times.g, 4.degree. C.). The supernatant
obtained after centrifugation was taken off and neutralized using a
solution consisting of 5M KOH, 1M TEA (adjusted pH 7.0) and then
centrifuged again (5 min at 16 000.times.g, 4.degree. C.). After
the end of the centrifugation, the supernatant was taken off, its
volume was determined and the amount of N-acetylated glucosamine
derivatives having a reducing end was determined using the method
described under b).
b) Determination of the Content of N-Acetylated Glucosamine
Derivatives Having Reducing Ends
[0214] 20 .mu.l of a solution consisting of 0.8M
K.sub.2B.sub.4O.sub.7, pH 9.6, are added to 100 .mu.l of the plant
extract obtained by the method described under a) and, after
thorough mixing, heated at 95.degree. C. for 5 min. After cooling
of the mixture to room temperature, 0.7 ml of Ehrlich's reagent
(solution consisting of 10 g of DMAB in 12.5 ml of conc. HCl, 87.5
ml of glacial acetic acid, 1:10 diluted with glacial acetic acid)
is added to the mixture, which is mixed again and incubated at
37.degree. C. for a further 30 minutes. The mixture is then
centrifuged at 16 000.times.g for 1 minute, and the optical density
(OD) of the supernatant obtained after centrifugation is
subsequently determined in a photometer at 585 nm.
c) Calculation of the Concentration of N-Acetylated Glucosamine
Derivatives
[0215] First, a calibration curve was established using defined
amounts of N-acetylglucosamine 6-phosphate. To this end, the OD of
solutions comprising 0 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM and 10 mM of
N-acetylglucosamine 6-phosphate was determined according to the
method described under b).
[0216] The calibration curve was established in Microsoft Excel by
fitting a second order polynomic trend/regression line of the
formula y=ax.sup.2+bx+c or y=x.sup.2+px+q to the points measured
for the individual concentrations. To calculate the values, the
equation obtained was resolved for x, resulting in: x=-p/2-square
root (p.sup.2/4-q), where p=b/a, q=(c-y)/a and y is the measured OD
of the unknown sample. Taking into account the fresh weight
employed, the volume used and taking into account any dilution
factor used, the contents were calculated in .mu.mol (of the
solution measured) or in .mu.mol per g of fresh weight.
5. Isolation of Glucosaminoglycans from Plant Tissue Using the
Example of Hyaluronan
[0217] To detect the presence of hyaluronan and to determine the
hyaluronan content in plant tissue, plant material was worked up as
follows: 200 .mu.l of water (demineralized, conductivity=18
M.OMEGA.) were added to about 0.3 g of leaf or tuber material, and
the mixture was comminuted in a laboratory oscillating ball mill
(MM200, from Retsch, Germany) (30 sec at 30 Hz). A further 800
.mu.l of water (demineralized, conductivity=18 M.OMEGA.) were then
added, and the mixture was mixed well (using, for example, a Vortex
mixer). Cell debris and insoluble components were separated from
the supernatant by centrifuging at 16 000.times.g for 5 minutes. An
aliquot of the supernatant obtained was used to determine the
amount of hyaluronan.
[0218] In the case of tomato fruits, in each case a whole ripe
tomato fruit was worked up. To this end, the weight of the tomato
fruit was determined, the tomato was comminuted in a Warring
blender with a little water, the comminuted sample was freed from
cell debris by centrifugation at 3600.times.g for 30 minutes and
the volume of the extract was determined. An aliquot of the
supernatant obtained was used to determine the amount of
hyaluronan.
6. Purification of Glucosaminoglycans Using the Example of
Hyaluronan
[0219] After addition of 100 ml of water (demineralized,
conductivity=18 M.OMEGA.), about 100 grams of plant material were
comminuted in a Warring blender at maximum speed for about 30
seconds. If relatively large parts of plants, such as, for example,
tubers or tomato fruits, were used for isolation, they were cut
beforehand into pieces of a size of about 1 cm.sup.3. The cell
debris was then removed using a tea sieve. The cell debris which
had been separated off was once more suspended in 300 ml of water
(demineralized, conductivity=18 M.OMEGA.) and again removed using a
tea sieve. The two suspensions obtained (100 ml+300 ml) were
combined and centrifuged at 13 000.times.g for 15 minutes. NaCl was
added to the centrifugation supernatant obtained until a final
concentration of 1% had been reached. After the NaCl had gone into
solution, precipitation was carried out by addition of twice the
volume of ethanol followed by thorough mixing and incubation at
-20.degree. C. overnight. The mixture was then centrifuged at 13
000.times.g for 15 minutes. The sedimented precipitate obtained
after this centrifugation was dissolved in 100 ml of buffer (50 mM
TrisHCl, pH 8, 1 mM CaCl.sub.2) and proteinase K was then added to
a final concentration of 100 .mu.g/ml and the solution was
incubated at 42.degree. C. for 2 hours. This was followed by 10
minutes of incubation at 95.degree. C. Once more, NaCl was added to
this solution until a final concentration of 1% had been reached.
After the NaCl had gone into solution, another precipitation was
carried out by addition of twice the volume of ethanol, thorough
mixing and incubation at -20.degree. C. for about 96 hours. This
was followed by 15 minutes of centrifugation at 13 000.times.g. The
sedimented precipitate obtained after this centrifugation was
dissolved in 30 ml of water (demineralized, conductivity=18
M.OMEGA.), and once more, NaCl was added to a final concentration
of 1%. By adding twice the volume of ethanol, thorough mixing and
incubation at -20.degree. C. overnight, another precipitation was
carried out. The precipitate obtained after subsequent
centrifugation at 13 000.times.g for 15 minutes was dissolved in 20
ml of water (demineralized, conductivity=18 M.OMEGA.).
[0220] Further purification was carried out by centrifugal
filtration. To this end, in each case 5 ml of the dissolved
precipitate were applied to a membrane filter (CentriconAmicon,
pore width 10 000 NMWL, Prod. No. UCF8 010 96), and the sample was
centrifuged at 2200.times.g until only about 3 ml of the solution
above the filter remained. Two more times, in each case 3 ml of
water (demineralized, conductivity=18 M.OMEGA.) were then added to
the solution above the membrane and in each case re-centrifuged
under identical conditions until, at the end, only about 3 ml of
the solution above the filter remained. The solutions still present
above the membrane after centrifugal filtration were taken off, and
the membrane was rinsed repeatedly (three to five times) with about
1.5 ml of water (demineralized, conductivity=18 M.OMEGA.). All
solutions which were still present above the membrane and the
solutions obtained from rinsing were combined, NaCl was added to a
final concentration of 1%, after the NaCl had gone into solution,
twice the volume of ethanol was added, the sample was mixed and a
precipitate was obtained by storage at -20.degree. C. overnight.
The precipitate obtained after subsequent centrifugation at 13
000.times.g for 15 minutes was dissolved in 4 ml of water
(demineralized, conductivity=18 M.OMEGA.) and then freeze-dried (24
hours under a pressure of 0.37 mbar, freeze drying apparatus Christ
Alpha 1-4, from Christ, Osterode, Germany).
7. Detection of Hyaluronan and Determination of the Hyaluronan
Content
[0221] Hyaluronan was detected using a commercial test (Hyaluronic
Acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod. No.
029-001) according to the instructions of the manufacturer which
are herewith incorporated into the description by way of reference.
The test principle is based on the availability of a protein which
binds specifically to hyaluronan (HABP) and is carried out
similarly to an ELISA, where a color reaction indicates the
hyaluronan content in the sample examined. Accordingly, for the
quantitative determination of hyaluronan, the samples to be
measured should be employed in a concentration such that it is
within the stated limits (for example: dilution of the sample in
question or use of less water for extracting hyaluronan from plant
tissue, depending on whether a limit was exceeded or not
reached).
8. Determination of the Activity of a GFAT
[0222] The activity of a protein having the activity of a GFAT is
determined as described in Rachel et al. (1996, J. Bacteriol. 178
(8), 2320-2327).
[0223] To distinguish whether a protein has the activity of a
GFAT-1 or GFAT-2, the method described in Hu et al. (2004, J. Biol.
Chem. 279 (29), 29988-29993) is used.
9. Detection of N-Acetylated Glucosamine Derivatives by Mass
Spectroscopy
[0224] To detect N-acetylated glucosamine derivatives by mass
spectroscopy, plant tissue was worked up as under General Methods
Item 4 a). To obtain an extract as free of salt as possible, the
respective samples were, prior to the examination by mass
spectroscopy, initially frozen at -20.degree. C. and thawed during
centrifugation (16 000.times.g at room temperature). For the
measurement, the supernatant was diluted 1:20 with a methanol:water
mixture in a ratio of 1:1 (volume/volume).
[0225] To increase the detection sensitivity for weak signals
(peaks), MS spectra with three different detector sensitivities
were recorded. However, in this case the response of the detector
is no longer linear, which is noted when the signal intensities
(peak areas) of different metabolites are compared and which should
be taken into account. To ensure that the measurements can be
compared with one another, it was ensured that the individual
samples gave identical signal intensities (in cps, counts per
second) at the same detector setting.
[0226] The areas of the resulting signals (peak areas) assigned to
the different metabolites are stated relatively to the peak area of
hexoses (m/z=179) in %. The ratio of the signal intensities (peak
areas) in different samples can be used to infer the concentration
ratios of the corresponding N-acetylated glucosamine derivatives in
relation to the concentration of hexoses in the sample in
question.
[0227] MS-MS measurements of the individual samples and of
individual corresponding reference substances (glucosamine,
N-acetyl glucosamine, glucosamine 6-posphate glucosamine
1-phosphate, N-acetylglucosamine 6-phosphate, N-acetylglucosamine
1-phosphate, UDP-N-acetylglucosamine) were carried out in parallel.
In this way, it is possible to assess whether the signal (peak)
used for determining the area is a signal generated exclusively by
a specific metabolite or by specific isomeric metabolites having
the same mass, or whether the signal in question can be assigned
only partially to the corresponding metabolite or the corresponding
specific isomeric metabolites having the same mass.
[0228] MS and MS-MS spectra were recorded in the negative mode
using a Q-STAR Pulsar i hybrid mass spectrometer from Applied
Biosystems fitted with a nano-electrospray source. The ions
detected were mainly deprotonated ions with a single charge.
[0229] The measurements were carried out under the following
conditions:
TABLE-US-00001 Mass range 50-700 Da. Detector sensitivity: 2000,
2050 and 2100.
[0230] For each of the three detector settings, it was ensured that
the samples had similar signal intensities (in cps, counts per
second).
EXAMPLES
1. Acquisition of Nucleic Acid Sequences Coding for a Protein
Having the Activity of a GFAT-1 from the Mouse
[0231] The nucleic acid sequence coding for a protein having the
activity of a GFAT-1 (glutamine:fructose 6-phosphate
amidotransferase or glucosamine 6-phosphate synthase, EC 2.6.1.16)
was purchased from BioCat GmbH, Heidelberg, Germany (Art. No.
MMM1013-65346, cDNA clone MGC:58262, IMAGE:6742987). This is a
clone which is produced by the I.M.A.G.E. consortium
(http://image.llnl.gov) and distributed by BioCat GmbH. The cDNA
coding for a protein having the activity of a GFAT-1 was cloned
into the vector pCMV Sport 6 from Invitrogen. The plasmid was named
IC 365-256. The nucleic acid sequence coding for the protein having
the activity of a GFAT-1 from Mus musculus is shown under SEQ ID NO
4.
[0232] To facilitate subsequent cloning steps, the coding sequence
of the GFAT-1 was excised using Xho I and Eco RV from the plasmid
IC 365-256 and cloned into the plasmid pME9, which had been cut
with the same restriction endonucleases. The plasmid obtained was
named IC 367-256.
[0233] The plasmid pME9 is a pBlueSkript vector from Stratagene
(Prod. No. 212207) where, in contrast to the pBlueSkript vector
mentioned, pME9 contains a modified Multiple Cloning Site (MCS)
which, in addition to the MCS present in the pBlueSkript vector,
has an additional Pac I restriction site at both ends of the
MCS.
2. Acquisition of a Nucleic Acid Sequence Coding for a Protein
Having the Activity of a GFAT-2 from a Mouse
[0234] The nucleic acid sequence coding for a protein having the
activity of a GFAT-2 (glutamine:fructose 6-phosphate
amidotransferase or glucosamine 6-phosphate synthase, EC 2.6.1.16)
was purchased from Invitrogen (Clone ID 4167189, cDNA clone
MGC:18324, IMAGE:4167189). This is a clone produced by the
I.M.A.G.E. consortium (http://image.llnl.gov) and distributed by
Invitrogen. The cDNA coding for a protein having the activity of a
GFAT-2 was cloned into the vector pCMV Sport 6 from Invitrogen. The
plasmid was named IC 369-256. The nucleic acid sequence coding for
the protein having the activity of a GFAT-2 from Mus musculus is
shown under SEQ ID NO 6.
3. Synthesis of the Nucleic Acid Sequences Coding for a Protein
Having the Activity Of a Bacterial GFAT from Escherichia coli
[0235] The nucleic acid sequence coding for a protein having the
activity of a bacterial GFAT (glutamine:fructose 6-phosphate
amidotransferase or glucosamine 6-phosphate synthase, glms, EC
2.6.1.16) from Escherichia coli was synthesized by Entelechon GmbH
and cloned into the vector pCR4Topo from Invitrogen (Prod. No.
K4510-20). The plasmid obtained was named IC 373-256. The synthetic
nucleic acid sequence coding for the protein having the activity of
a bacterial GFAT from Escherichia coli is shown under SEQ ID NO 10.
The corresponding nucleic acid sequence originally isolated from
Escherichia coli is shown under SEQ ID NO 8.
4. Synthesis of Nucleic Acid Molecules Coding for a Hyaluronan
Synthase of Paramecium Bursaria Chlorella Virus 1
[0236] The nucleic acid sequence coding for a hyaluronan synthase
of Paramecium bursaria Chlorella Virus 1 was synthesized by
Medigenomix GmbH (Munich, Germany) and cloned into the vector
pCR2.1 from Invitrogen (Prod. No. K2000-01). The plasmid obtained
was named IC 323-215. The synthetic nucleic acid sequence coding
for the HAS protein from Paramecium bursaria Chlorella Virus 1 is
shown under SEQ ID NO 3. The corresponding nucleic acid sequence
originally isolated from Paramecium bursaria Chlorella Virus 1 is
shown under SEQ ID NO 1.
5. Synthesis of Nucleic Acid Molecules Coding for a Protein Having
the Activity of a UDP-Glucose Dehydrogenase of Paramecium Bursaria
Chlorella Virus 1
[0237] The nucleic acid sequence coding for a protein having the
activity of a UDP-glucose dehydrogenase from Paramecium bursaria
Chlorella Virus 1 was synthesized by Entelechon GmbH and cloned
into the vector pCR4Topo from Invitrogen (Prod. No. K4510-20). The
plasmid obtained was named IC 339-222. The synthetic nucleic acid
sequence coding for the protein having the activity of a
UDP-glucose dehydrogenase from Paramecium bursaria Chlorella Virus
1 is shown under SEQ ID NO 13. The corresponding nucleic acid
sequence originally isolated from Paramecium bursaria Chlorella
Virus 1 is shown under SEQ ID NO 11
6. Preparation of the Plant Expression Vector IR 47-71
[0238] The plasmid pBinAR is a derivative of the binary vector
plasmid pBin19 (Bevan, 1984, Nucl Acids Res 12: 8711-8721) which
was constructed as follows:
[0239] A 529 bp fragment comprising the nucleotides 6909-7437 of
the 35S promoter of the cauliflower mosaic virus was isolated as
EcoR I/Kpn I fragment from the plasmid pDH51 (Pietrzak et al, 1986
Nucleic Acids Res. 14, 5858) and ligated between the EcoR I and Kpn
I restriction sites of the polylinker of pUC18. This gave the
plasmid pUC18-35S. With the aid of the restriction endonucleases
Hind III and Pvu II, a 192 bp fragment comprising the
polyadenylation signal (3'-terminus) of the octopin synthase gene
(Gen 3) of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al, 1984,
EMBO Journal 3, 835-846) (nucleotides 11749-11939) was isolated
from the plasmid pAGV40 (Herrera-Estrella et al, 1983 Nature, 303,
209-213). After addition of Sph I linkers to the Pvu II restriction
site, the fragment was ligated between the Sph I and Hind III
restriction sites of pUC18-35S. This gave the plasmid pA7. From
this plasmid, the entire polylinker comprising the 35S promoter and
the OCS terminator was excised with EcoR I and Hind III and ligated
into the appropriately cut vector pBin19. This gave the plant
expression vector pBinAR (Hofgen and Willmitzer, 1990, Plant
Science 66, 221-230).
[0240] The promoter of the Patatin gene B33 from Solanum tuberosum
(Rocha-Sosa et al., 1989, EMBO J. 8, 23-29) was ligated as Dra I
fragment (nucleotides-1512-+14) into the vector pUC19, which had
been cut with Sst I and whose ends had been blunted with the aid of
T4-DNA polymerase. This gave the plasmid pUC19-B33. Using EcoR I
and Sma I, the B33 promoter was excised from this plasmid and
ligated into the appropriately cut vector pBinAR. This gave the
plant expression vector pBinB33.
[0241] To facilitate further cloning steps, the MCS (Multiple
Cloning Site) was widened. To this end, two complementary
oligonucleotides were synthesized, heated at 95.degree. C. for 5
minutes and slowly cooled to room temperature, and the
double-stranded fragment obtained was cloned into the Sal I and Kpn
I restriction sites of pBinB33. The oligonucleotides used for this
purpose had the following sequence:
TABLE-US-00002 5'-TCG ACA GGC CTG GAT CCT TAA TTA AAC TAG TCT CGA
GGA GCT CGG TAC-3' 5'-CGA GCT CCT CGA GAC TAG TTT AAT TAA GGA TCC
AGG CCT G-3'
[0242] The plasmid obtained was named IR 47-71.
7. Preparation of the Plant Expression Vector pBinARHyg
[0243] Using the restriction endonucleases EcoR I and Hind III, the
fragment comprising the 35S promoter, the OCS terminator and the
entire multiple cloning site was excised from the plasmid pA7 and
cloned into the vector pBIBHyg (Becker, 1990, Nucleic Acids Res.
18, 203) which had been cut with the same restriction
endonucleases. The plasmid obtained was named pBinARHyg.
8. Preparation of the Cloning Vector IC 317-204
[0244] Nucleic acid fragments comprising the OCS terminator were
isolated from the plasmid IR 47-71 using the restriction
endonucleases Xho I and Hind III and cloned into the vector
pBlueScript KS (from Stratagene, Prod. No. 212207), which had been
cut with the same restriction endonucleases. The plasmid obtained
was named IC 306-204.
[0245] Nucleic acid fragments comprising the B33 promoter were
isolated from the plasmid IR 47-71 using the restriction
endonucleases Bam HI and Eco RI and cloned into the vector
pBlueScript KS (from Stratagene, Prod. No. 212207), which had been
cut with the same restriction endonucleases. The plasmid obtained
was named IC 314-204.
[0246] From IC 306-204, the OCS terminator was isolated using the
restriction endonuclease Bam HI and cloned into the plasmid IC
314-204, which had been cut with the same restriction endonuclease.
The plasmid obtained was named IC 317-204.
9. Preparation of the Plant Expression Vector IC 341-222 Comprising
a Coding Nucleic Acid Sequence for a Hyaluronan Synthase of
Paramecium Bursaria Chlorella Virus 1
[0247] By restriction digestion with BamH I and Xho I, nucleic acid
molecules comprising the coding sequence of hyaluronan synthase
were isolated from the plasmid IC 323-215 and cloned into the BamH
I and Xho I restriction sites of the plasmid IR 47-71. The plant
expression vector obtained was named IC 341-222.
10. Preparation of the Plant Expression Vectors 349-222 Comprising
Coding Nucleic Acid Sequences for a Protein Having the Activity of
a UDP-Glucose Dehydrogenase from Paramecium Bursaria Chlorella
Virus 1
[0248] Using restriction digestion with BamH I and Kpn I, nucleic
acid molecules comprising the coding sequence for a protein having
the activity of a UDP-glucose dehydrogenase from Paramecium
bursaria Chlorella Virus 1 were isolated from the plasmid IC
339-222 and cloned into the plasmid pA7, which had been cut with
the same restriction endonucleases. The plasmid obtained was named
IC 342-222.
[0249] By restriction digestion with Xba I and Kpn I, nucleic acid
molecules comprising the coding sequence for a protein having the
activity a UDP-glucose dehydrogenase from Paramecium bursaria
Chlorella Virus 1 were isolated from the plasmid IC 342-222 and
cloned into the expression vector pBinAR Hyg, which had been cut
with Xba I and Kpn I. The plasmid obtained was named IC
349-222.
11. Preparation of the Plant Expression Vectors IC 376-271
Comprising Coding Nucleic Acid Sequences for a Protein Having the
Activity of a GFAT-1 from the Mouse and for a Protein Having the
Activity of a UDP-Glucose Dehydrogenase from Paramecium bursaria
Chlorella Virus 1
[0250] A nucleic acid fragment comprising the B33 promoter and the
OCS terminator, which fragment had been isolated from IC 317-204 by
restriction digestion using Eco RI, was cloned into the Eco RI
restriction site of the plasmid IC 349-222. Here, head-to-head
orientation of the promoters (25S and B33) was ensured. The vector
obtained was named IC 354-222.
[0251] To obtain a plant expression vector comprising a nucleic
acid sequence coding for a protein having the activity of a GFAT-1
from the mouse, the coding sequence of the protein having the
activity of a GFAT-1 from the mouse was isolated by restriction
digestion with Xho I and Eco RV from IC 365-256 and cloned into the
plasmid IC 354-222, which had been cut with Xho I and Ecl136 II.
The plant expression vector obtained was named IC 376-256.
12. Preparation of the Plant Expression Vector IC 372-256
Comprising Coding Nucleic Acid Sequences for a Protein Having the
Activity of a GFAT-2 from the Mouse and for a Protein Having the
Activity of a UDP-Glucose Dehydrogenase from Paramecium bursaria
Chlorella Virus 1
[0252] A nucleic acid fragment comprising the coding sequence of
the protein having the activity of a GFAT-2 from the mouse was
isolated from IC 369-256 by restriction digestion with Xho I and
Eco RV and cloned into the plasmid IC 354-222, which had been cut
with Xho I and Ecl136 II. The plant expression vector obtained was
named IC 372-256.
13. Preparation of the Plant Expression Vector 375-271 Comprising
Coding Nucleic Acid Sequences for a Protein Having the Activity of
a GFAT from Escherichia coli and for a Protein Having the Activity
of a UDP-Glucose Dehydrogenase from Paramecium bursaria Chlorella
Virus 1
[0253] A nucleic acid fragment comprising the coding sequence of
the protein having the activity of a GFAT from Escherichia coli was
isolated from IC 373-256 by restriction digestion with Xho I and
Eco RV and cloned into the plasmid IC 354-222, which had been cut
with Xho I and Ecl136 II. The plant expression vector obtained was
named IC 375-271.
14. Preparation of the Plant Expression Vector IC 398-311
Comprising a Coding Nucleic Acid Sequence for a Protein Having the
Activity of a GFAT from Escherichia coli
[0254] By restriction digestion with Ecl 136 I and Xho I, the
coding sequence of the protein having the activity of a bacterial
GFAT from E. coli was isolated from the plasmid IC 373-256 and
ligated into the Sma I and Sal I restriction sites of the vector
pBinAR Hyg. The plant expression vector obtained was named IC
398-311.
15. Preparation of the Plant Expression Vector IC 386-299
[0255] By PCR using genomic DNA isolated from leaves of rice (Oryza
sativa, cultivar M202) using DNA polymerase (Expand High Fidelity
PCR Systems, Roche Prod. No.: 1732641), the DNA of the prolamin
promoter from rice (EMBL Accession NO D63901, Sha et al., 1996,
Biosci. Biotech. Biochem. 60, 335-337, Wu et al., 1998. Plant Cell
Physiol. 39(8), 885-889) was isolated. The amplicon obtained from
this PCR reaction was cloned into the vector pCR 2.1 using the TA
cloning kit (Invitrogen Prod. No.: KNM2040-01). The plasmid
obtained was named MI 4-154. Conditions used for the amplification
of the DNA coding for the prolamin promoter:
[0256] The conditions and buffers stated by the manufacturer and 50
ng of total DNA were used.
TABLE-US-00003 0.83 .mu.M dNTP mix 0.25 .mu.M Primer prol-F1
5'-AAAAACTAGTTCTACATCGGCTTAGGTGTAGCAACACG 0.25 .mu.M primer prol-R1
5'-AAAAGATATCTGTTGTTGGATTCTACTACTATGCTTCAA
[0257] Reaction conditions:
TABLE-US-00004 Step 1 94.degree. C. 15 sec Step 2 60.degree. C. 15
sec Step 3 72.degree. C. 45 sec
[0258] First, the reaction according to steps 1 to 3 was carried
out using 35 repetitions (cycles). After the reaction had ended,
the reaction mixture was cooled to 4.degree. C. Subsequent cloning
into the vector pCR 2.1 using the TA cloning kit (Invitrogen Prod.
No.: KNM2040-01) was carried out following the conditions stated by
the manufacturer. The plasmid comprising the prolamin promoter from
rice was named MI 4-154.
[0259] A nucleic acid fragment comprising the coding sequence of
the protein having the activity of a GFAT-2 from the mouse was
isolated by restriction digestion using the restriction
endonucleases Not I and Kpn I from the plasmid IC 369-256 and
cloned into the vector pMCS5 (purchased from MoBiTec), which had
been digested with Not I and Kpn I. The plasmid obtained was named
IC 385-299. In the next step, the nucleic acid fragment comprising
the coding sequence of the protein having the activity of a GFAT-2
from the mouse was isolated by restriction digestion with the
restriction endonucleases Xho I and Hpa I from IC 385-299 and
cloned into the plasmid MI 9-154, which had been cut with Xho I and
Ecl136 II. The plant expression vector obtained was named IC
386-299. Starting vector for the preparation of the vector MI 9-154
is the plasmid ML 18-56 (WO 05/030941). An MCS synthesized by two
oligonucleotides and having the appropriate sticky ends and
comprising the restriction sites Pst I, Sac I, Bln I, Xho I, Hpa I,
Spe I and Hind III was introduced into the plasmid ML 18-56, which
had been digested with Hind III and Pst I. The vector obtained was
named MI 8-154.
[0260] By digestion with Eco RV and Spe I, the prolamin promoter
was isolated from MI 4-154 and ligated into the vector MI 8-154,
which had been digested with Hpa I and Spe I. The vector obtained
was named MI 9-154.
16. Potato Plants Comprising a Nucleic Acid Molecule Coding for a
Protein Having the Activity of a Bacterial GFAT
a) Transformation of Potato Plants
[0261] Potato plants (cultivar Desiree) were transformed by the
method given in General Methods Item 1 using the plant expression
vector IC 398-311, which comprises a coding nucleic acid sequence
for a protein having the activity of a bacterial GFAT from
Escherichia coli under the control of the promoter of the patatin
gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J.
8, 23-29). The transgenic lines obtained, which are transformed
with the plasmid IC 398-311, were named 432 ES.
b) Analysis of Lines 432 ES
[0262] Plants of the line 432 ES were cultivated in a greenhouse in
soil in 6 cm pots. In each case about 0.3 g to 0.8 g of leaf
material, harvested from individual plants, was worked up according
to the method described under General Methods Item 4, and the
content of N-acetylated glucosamine derivatives was determined. For
individual plants having an increased content of N-acetylated
glucosamine derivatives, the following results were obtained:
TABLE-US-00005 TABLE 1 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in
leaves of independent transgenic plants of the line 432 ES. Plant
.mu.mol/g FW 432ES 1 2.97 432ES 2 0.51 432ES 4 2.19 432ES 5 3.99
432ES 6 6.20 432ES 7 2.98 432ES 8 0.48 432ES 9 11.48 432ES 10 0.30
432ES 11 6.89 432ES 12 5.45 432ES 13 0.23 432ES 14 0.80 432ES 15
1.75 432ES 16 4.87 432ES 18 3.38 432ES 19 6.38 432ES 21 1.42 432ES
22 9.73 432ES 23 5.88 432ES 25 4.45 432ES 26 1.81 432ES 27 1.75
432ES 28 0.45 432ES 32 4.56 432ES 33 3.64 432ES 35 3.64 432ES 37
6.67 432ES 38 0.95 432ES 40 8.69 432ES 42 1.47 432ES 43 5.41 432ES
44 6.33 432ES 45 3.39 wt 1 0.05 wt 2 0.26 wt 3 0.17 Column 1 refers
in each case to the plant, independently obtained from the
transformation, from which the material was harvested ("wt" refers
to plants which have not been transformed).
[0263] These results show that plants having a foreign nucleic acid
molecule coding for a protein having the activity of a bacterial
GFAT have a considerably higher content of N-acetylated glucosamine
derivatives than correspondingly non-transformed wild-type
plants.
17. Rice Plants Comprising a Nucleic Acid Molecule Coding for a
Protein Having the Activity of a GFAT-2
a) Transformation of Rice Plants
[0264] Rice plants (variety M202) were transformed according to the
method given under General Methods Item 3 with the plant expression
vector IC 386-299, which comprises a coding nucleic acid sequence
for a protein having the activity of a GFAT-2 from the mouse under
the control of the promoter of the 13 kDa prolamin polypeptide. The
transgenic lines obtained, which are transformed with the plasmid
IC 386-299, were named GAOS0788.
b) Analysis of the Lines GAOS0788
[0265] Independent plants, originating from the transformation with
the plasmid IC 386-299, of the line GAOS0788 were cultivated in
soil in a greenhouse. From each plant, about 20-25 ripe seeds
(grains) were harvested, the husks were removed with a dehusker
(Laboratory Paddy sheller, Grainman, Miami, Fla., USA) and about 7
brown rice seeds (pools) of each line were comminuted in a
laboratory oscillating ball mill (MM200, from Retsch, Germany, 30
sec at 30 Hz), resulting in a flour. Using the method described
under General Methods Item 4, the content of N-acetylated
glucosamine derivatives was then determined. For individual plants
having an increased content of N-acetylated glucosamine
derivatives, the following results were obtained:
TABLE-US-00006 TABLE 2 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in pools
of ripe seeds of independent transqenic plants of the line
GAOS0788. Plant .mu.mol/g FW GAOS0788-00101 14.50 GAOS0788-00202
17.36 GAOS0788-00301 14.46 GAOS0788-00501 23.07 GAOS0788-00602 7.75
GAOS0788-00701 4.44 GAOS0788-00802 17.43 GAOS0788-00901 10.13
GAOS0788-01001 6.38 GAOS0788-01202 8.32 GAOS0788-01401 8.64
GAOS0788-01502 2.97 GAOS0788-01602 8.15 GAOS0788-01701 16.50
GAOS0788-02002 5.65 GAOS0788-02202 5.15 GAOS0788-02301 7.82
GAOS0788-02401 20.89 GAOS0788-02501 6.67 GAOS0788-02601 7.34
GAOS0788-02701 4.31 GAOS0788-02802 8.02 GAOS0788-02901 4.74
GAOS0788-03001 4.36 GAOS0788-03101 11.83 GAOS0788-03202 2.76
GAOS0788-03302 12.82 Control n.d. Control n.d. Column 1 refers to
the plant, independently obtained from the transformation, from
which material was harvested (here, "control" refers to plants
transformed with a plasmid having no nucleic acid molecule coding
for a protein having the activity of a GFAT. Non-detectable amounts
are marked "n.d.".
c) Analysis of Individual Seeds of the Plants GAOS0788-02401 and
GAOS0788-00501
[0266] The seeds harvested in Example b) originated from plants
obtained directly after transformation, which plants were thus
heterozygotic with respect to the respective integration sites of
the T-DNAs in question. Accordingly, as a result of Mendel's laws
of inheritance, the seed pools analyzed contained seeds comprising
various amounts of the T-DNAs in question, it also being possible
for individual seeds not having any T-DNAs integrated by
transformation to be present in the respective pools. Thus, single,
individual brown seeds from the plants of the line GAOS0788-02401
and plants of the line GAOS0788-00501 were each examined by the
method described under General Methods Item 4 for their content of
N-acetylated glucosamine derivatives. The following results were
obtained:
TABLE-US-00007 TABLE 3 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) of individual
seeds of the plants from lines GAOS0788-02401 and GAOS0788-00501.
Sample .mu.mol/g FW GAOS0788-02401 seed 1 n.d GAOS0788-02401 seed 2
22.41 GAOS0788-02401 seed 3 38.47 GAOS0788-02401 seed 4 16.57
GAOS0788-02401 seed 5 17.67 GAOS0788-02401 seed 6 3.79
GAOS0788-02401 seed 7 10.14 GAOS0788-02401 seed 8 18.70
GAOS0788-00501 seed 1 n.d GAOS0788-00501 seed 2 17.20
GAOS0788-00501 seed 3 19.89 GAOS0788-00501 seed 4 15.47
GAOS0788-00501 seed 5 9.31 GAOS0788-00501 seed 6 20.88
GAOS0788-00501 seed 7 25.31 GAOS0788-00501 seed 8 31.92
GAOS0788-00501 seed 9 28.82 GAOS0788-00501 seed 10 43.35 Control
seed 1 n.d Control seed 2 n.d Control seed 3 n.d Control seed 4 n.d
In each case, column 1 refers to the plant, independently obtained
from the transformation, from which individual seeds were harvested
and analyzed (here, "control" refers to seeds of plants transformed
with a construct comprising no nucleic acid molecule coding for a
protein having the activity of a GFAT). Non-detectable amounts are
marked "n.d.".
[0267] The results obtained show that flours from seeds (grains) of
rice plants having a nucleic acid molecule coding for a protein
having the activity of a GFAT-2 have a considerably higher content
of N-acetylated glucosamine derivatives compared to flours produced
from plants having no nucleic acid molecule coding for a protein
having the activity of a GFAT-2.
18. Synthesis of N-Acetylated Glucosamine Derivatives in Tomato
Plants Transformed with Nucleic Acid Molecules Coding for Various
Isoforms of a Protein Having the Activity of a GFAT
a) Production of Tomato Plants Comprising a Foreign Nucleic Acid
Molecule Coding For a Protein Having the Activity of a GFAT-1
[0268] Tomato plants (cultivar Moneymaker) were transformed by the
method given under General Methods Item 2 with the plant expression
vector IC 376-271, which comprises a coding nucleic acid sequence
for a protein having the activity of a UDP-glucose dehydrogenase
and a foreign nucleic acid molecule coding for a protein having the
activity of a GFAT-1. The transgenic lines obtained, which are
transformed with the plasmid 376-271, were named 420 ES. Proteins
having the activity of a UDP-glucose dehydrogenase catalyze the
synthesis of UDP-GlcA from UDP-glucose. In addition to GlcNAc, some
glucosaminoglycan synthases, such as, for example, hyaluronan
synthase, require UDP-GlcA as substrate.
b) Analysis of the Lines 420 ES
[0269] Plants of the line 420 ES were cultivated in hydroculture in
pots in a greenhouse. In each case about 5 g of plant material,
harvested from individual plants, were worked up using the method
described under General Methods Item 4, and the content of
N-acetylated glucosamine derivatives was determined. Here, per
plant, a plurality of independent measurements were carried out for
each worked-up sample. The following results were obtained for
individual plants:
TABLE-US-00008 TABLE 4 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in
leaves of independent transgenic plants of the line 420 ES.
.mu.mol/g Mean Plant FW [.mu.mol/g FW] 420ES 1 a 0.15 0.15 420ES 1
b 0.10 420ES 1 c 0.15 420ES 2 a 0.11 0.10 420ES 2 b 0.09 420ES 2 c
0.10 420ES 3 a 0.29 0.28 420ES 3 b 0.30 420ES 3 c 0.25 420ES 4 a
0.20 0.18 420ES 4 b 0.19 420ES 4 c 0.16 420ES 5 a 0.10 0.09 420ES 5
b 0.08 420ES 5 c 0.09 420ES 6 a 0.24 0.27 420ES 6 c 0.29 420ES 7 b
1.12 1.31 420ES 7 c 1.50 420ES 8 a 0.05 0.06 420ES 8 b 0.05 420ES 8
c 0.06 420ES 9 a 0.02 0.02 420ES 9 b 0.01 420ES 9 c 0.02 420ES 10 a
0.05 0.04 420ES 10 b 0.03 420ES 10 c 0.05 420ES 11 a 0.06 0.06
420ES 11 b 0.10 420ES 11 c 0.03 420ES 12 a 0.09 0.08 420ES 12 b
0.06 420ES 13 a 0.02 0.02 420ES 13 b 0.01 420ES 13 c 0.03 420ES 14
a 0.02 0.03 420ES 14 b 0.04 420ES 14 c 0.04 420ES 15 a 0.05 0.06
420ES 15 b 0.06 420ES 15 c 0.06 420ES 16 a 0.08 0.07 420ES 16 b
0.06 420ES 16 c 0.07 420ES 17 a 0.08 0.07 420ES 17 b 0.07 420ES 17
c 0.08 420ES 18 a 0.07 0.08 420ES 18 b 0.09 420ES 18 c 0.09 420ES
19 a 0.03 0.03 420ES 19 b 0.00 420ES 19 c 0.05 420ES 20 a 0.04 0.06
420ES 20 b 0.07 420ES 20 c 0.05 420ES 22 a 0.08 0.08 420ES 22 b
0.07 420ES 22 c 0.08 420ES 23 a 0.14 0.13 420ES 23 b 0.11 420ES 23
c 0.13 420ES 24 a 0.05 0.05 420ES 24 b 0.04 420ES 24 c 0.05 420ES
25 a 0.05 0.06 420ES 25 b 0.07 420ES 25 c 0.06 420ES 26 a 0.13 0.09
420ES 26 b 0.06 420ES 26 c 0.08 420ES 27 a 0.09 0.08 420ES 27 b
0.10 420ES 27 c 0.05 420ES 28 a 0.01 0.01 420ES 28 b 0.02 420ES 28
c 0.01 420ES 29 a 0.09 0.08 420ES 29 b 0.07 420ES 29 c 0.07 420ES
30 a 0.04 0.03 420ES 30 b 0.03 420ES 30 c 0.01 wt 7 a 0.09 0.10 wt
7 b 0.11 wt 7 c 0.09 wt 12 a 0.02 0.01 wt 12 b n.d wt 12 c 0.03
Column 1 refers to the plant, independently originating from the
transformation, from which the material was harvested (here, "wt"
refers to non-transformed plants). The extension of the names of
the plants by a, b or c denotes independent measurements carried
out for the worked-up sample in question. Non-detectable amounts
are marked "n.d.".
[0270] These results show that plants having a foreign nucleic acid
molecule coding for a protein having the activity of a GFAT-1 and
coding for a protein having the activity of a UDP-glucose
dehydrogenase have a content of N-acetylated glucosamine
derivatives which is slightly higher than that of corresponding
non-transformed wild-type plants.
c) Production of Tomato Plants Comprising a Foreign Nucleic Acid
Molecule Coding for a Protein Having the Activity of a GFAT-2
[0271] Tomato plants (cultivar Moneymaker) were transformed by the
method given under General Methods Item 2 with the plant expression
vector IC 372-256 comprising a coding nucleic acid sequence for a
protein having the activity of a UDP-glucose dehydrogenase and a
foreign nucleic acid molecule coding for a protein having the
activity of a GFAT-2. The transgenic lines obtained, which are
transformed with the plasmid IC 372-256, were named 421 ES.
d) Analysis of Lines 421 ES
[0272] Plants of the line 421 ES were cultivated in hydroculture in
pots in a greenhouse. In each case about 5 g of plant material,
harvested from individual plants, were worked up using the method
described under General Methods Item 4, and the content of
N-acetylated glucosamine derivatives was determined. Here, per
plant, a plurality of independent measurements were carried out for
each worked-up sample. The following results were obtained for
individual plants:
TABLE-US-00009 TABLE 5 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in
leaves of independent transgenic plants of the line 421 ES.
.mu.mol/g Mean Plant FW [.mu.mol/g FW] 421ES 1 a 0.60 0.67 421ES 1
b 0.61 421ES 1 c 0.80 421ES 3 a 1.07 1.10 421ES 3 b 1.07 421ES 3 c
1.17 421ES 4 a 2.22 2.00 421ES 4 b 1.88 421ES 4 c 1.89 421ES 5 a
0.79 0.87 421ES 5 b 1.07 421ES 5 c 0.74 421ES 6 a 0.62 0.74 421ES 6
b 0.76 421ES 6 c 0.85 421ES 7 a 1.20 1.01 421ES 7 b 1.01 421ES 7 c
0.84 421ES 9 a 0.35 0.40 421ES 9 b 0.36 421ES 9 c 0.48 421ES 10 a
0.08 0.16 421ES 10 b 0.18 421ES 10 c 0.22 421ES 11 a 2.96 2.78
421ES 11 b 2.61 421ES 11 c 2.78 421ES 12 a 1.13 0.96 421ES 12 b
0.82 421ES 12 c 0.93 421ES 19 a 0.04 0.04 421ES 19 b 0.03 421ES 21
a 0.21 0.25 421ES 21 b 0.36 421ES 21 c 0.19 421ES 23 a 0.01 0.01
421ES 23 b 0.01 421ES 23 c 0.02 421ES 26 a 0.18 0.16 421ES 26 b
0.19 421ES 26 c 0.10 421ES 27 a 0.11 0.13 421ES 27 b 0.16 421ES 27
c 0.12 421ES 28 a 0.02 0.01 421ES 28 b n.d. 421ES 28 c 0.01 421ES
29 a 0.35 0.40 421ES 29 b 0.46 421ES 29 c 0.39 421ES 31 a 0.14 0.14
421ES 31 b 0.16 421ES 31 c 0.11 421ES 32 a 0.04 0.03 421ES 32 b
0.01 421ES 32 c 0.05 421ES 33 a 0.12 0.11 421ES 33 b 0.08 421ES 33
c 0.13 421ES 34 a 0.32 0.34 421ES 34 b 0.37 421ES 34 c 0.34 421ES
35 a 0.20 0.21 421ES 35 b 0.24 421ES 35 c 0.17 421ES 36 a 0.07 0.06
421ES 36 b 0.07 421ES 36 c 0.03 421ES 37 a 0.12 0.12 421ES 37 b
0.11 421ES 37 c 0.14 421ES 38 a 0.32 0.34 421ES 38 b 0.34 421ES 38
c 0.37 wt 8 a n.d. n.d. wt 8 c n.d. wt 13 a n.d. n.d. wt 13 b n.d.
wt 13 c n.d. Column 1 refers to the plant, independently
originating from the transformation, from which the material was
harvested (here, "wt" refers to non-transformed plants). The
extension of the names of the plants by a, b or c denotes
independent measurements carried out for the worked-up sample in
question. Non-detectable amounts are marked "n.d.".
[0273] These results show that plants having a foreign nucleic acid
molecule coding for a protein having the activity of a GFAT-2 and
coding for a protein having the activity of a UDP-glucose
dehydrogenase have a content of N-acetylated glucosamine
derivatives which is considerably higher than that of
correspondingly non-transformed wild-type plants.
e) Production of Tomato Plants Comprising a Foreign Nucleic Acid
Molecule Coding for a Protein Having the Activity of a Bacterial
GFAT
[0274] Tomato plants (cultivar Moneymaker) were transformed by the
method given under General Methods Item 2 with the plant expression
vector IC 375-271 comprising a coding nucleic acid sequence for a
protein having the activity of a UDP-glucose dehydrogenase and a
foreign nucleic acid molecule coding for a protein having the
activity of a bacterial GFAT. The transgenic lines obtained, which
are transformed with the plasmid IC 375-271, were named 422 ES.
f) Analysis of Lines 422 ES
[0275] Plants of the line 422 ES were cultivated in hydroculture in
pots in a greenhouse. In each case about 5 g of plant material,
harvested from individual plants, were worked up using the method
described under General Methods Item 4, and the content of
N-acetylated glucosamine derivatives was determined. Here, per
plant, a plurality of independent measurements were carried out for
each worked-up sample. The following results were obtained for
individual plants:
TABLE-US-00010 TABLE 6 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in
leaves of independent transgenic plants of the line 422 ES.
.mu.mol/g Mean Plant FW [.mu.mol/g FW] 422ES 2 a 13.96 14.50 422ES
2 b 13.39 422ES 2 c 16.13 422ES 3 a 0.28 0.29 422ES 3 b 0.29 422ES
3 c 0.30 422ES 4 a 0.20 0.18 422ES 4 b 0.13 422ES 4 c 0.21 422ES 5
a 10.57 9.97 422ES 5 b 9.74 422ES 5 c 9.60 422ES 6 a 16.58 16.20
422ES 6 b 16.11 422ES 6 c 15.91 422ES 7 a 3.13 2.99 422ES 7 b 2.64
422ES 7 c 3.19 422ES 8 a 16.50 14.70 422ES 8 b 14.32 422ES 8 c
13.27 422ES 9 a 9.76 9.72 422ES 9 b 9.33 422ES 9 c 10.07 422ES 11 a
5.80 5.40 422ES 11 b 5.34 422ES 11 c 5.05 422ES 12 a 11.57 12.23
422ES 12 b 11.65 422ES 12 c 13.46 422ES 13 a 13.11 10.89 422ES 13 b
10.54 422ES 13 c 9.02 422ES 14 a 7.68 7.75 422ES 14 b 8.05 422ES 14
c 7.52 422ES 16 a 14.02 14.45 422ES 16 b 13.35 422ES 16 c 15.98
422ES 17 a 10.79 9.72 422ES 17 b 9.99 422ES 17 c 8.37 422ES 18 a
3.09 4.20 422ES 18 b 4.55 422ES 18 c 4.96 422ES 19 a 6.43 5.99
422ES 19 b 4.94 422ES 19 c 6.59 422ES 20 a 15.85 15.50 422ES 20 b
15.87 422ES 20 c 14.79 422ES 21 a 0.32 0.35 422ES 21 c 0.38 wt 9 a
0.36 0.23 wt 9 b 0.19 wt 9 c 0.13 wt 14 a n.d. n.d. wt 14 b n.d. wt
14 c n.d. Column 1 refers to the plant, independently originating
from the transformation, from which the material was harvested
(here, "wt" refers to non-transformed plants). The extension of the
names of the plants by a, b or c denotes independent measurements
carried out for the worked-up sample in question. Non-detectable
amounts are marked "n.d.".
g) Analysis of Fruits of Lines 420 ES, 421 ES and 422 ES
[0276] Ripe fruits were harvested from selected plants of lines 420
ES, 421 ES and 422 ES. Various whole tomato fruits of individual
plants were harvested and worked up using the method described
under General Methods Item 4, and the content of N-acetylated
glucosamine derivatives was determined. Here, independent
measurements were carried out for different fruits of a plant. The
following results were obtained for individual plants:
TABLE-US-00011 TABLE 7 Amount of N-acetylated glucosamine
derivatives (in .mu.mol per gram of fresh weight) measured in
fruits of independent transgenic plants of lines 420 ES, 421 ES and
422 ES. .mu.mol/g Mean Plant FW [.mu.mol/g FW] 420ES 2 I 0.01 0.01
420ES 2 II 0.01 420ES 3 I 0.07 0.06 420ES 3 II 0.06 420ES 4 I 0.04
0.04 420ES 4 II n.d 420ES 4 III 0.04 420ES 6 I 0.09 0.05 420ES 6 II
0.03 420ES 6 III 0.04 420ES 7 I 0.01 0.03 420ES 7 II 0.04 420ES 7
III 0.05 420ES 7 IV 0.03 420ES 8 I 0.03 0.04 420ES 8 II 0.04 420ES
8 III 0.04 420ES 12 I 0.00 0.04 420ES 12 II 0.07 420ES 17 I 0.05
0.05 420ES 17 II 0.06 420ES wt 7 I n.d 0.03 420ES wt 7 II 0.04
420ES wt 7 III 0.03 421ES 4 I 0.94 0.86 421ES 4 II 0.85 421ES 4 III
0.67 421ES 4 IV 0.79 421ES 4 V 1.02 421ES 5 I 0.35 0.53 421ES 5 II
0.72 421ES 5 III 0.67 421ES 5 IV 0.45 421ES 5 V 0.48 421ES 21 I
2.02 1.17 421ES 21 II 0.92 421ES 21 III 0.96 421ES 21 IV 0.79 421ES
25 I 0.61 0.76 421ES 25 II 0.75 421ES 25 III 0.91 421ES 27 I 0.86
0.89 421ES 27 II 0.91 421ES 27 III 0.90 421ES 29 I 0.48 0.76 421ES
29 II 0.52 421ES 29 III 0.52 421ES 29 IV 1.53 421ES 33 I 0.74 0.67
421ES 33 II 0.83 421ES 33 III 0.45 421ES 35 I 0.48 0.77 421ES 35 II
0.79 421ES 35 III 0.87 421ES 35 IV 0.95 421ES 38 I 0.97 1.21 421ES
38 II 1.35 421ES 38 III 1.29 421ES wt 13 I 0.03 0.04 421ES wt 13 II
0.05 421ES wt 13 III n.d 422ES 2 I 3.17 4.26 422ES 2 II 3.74 422ES
2 III 5.79 422ES 2 IV 4.90 422ES 2 V 3.73 422ES 5 I 2.12 3.41 422ES
5 II 1.76 422ES 5 III 1.99 422ES 5 IV 3.26 422ES 5 V 5.27 422ES 5
VI 4.49 422ES 5 VII 4.95 422ES 6 I 7.41 7.41 422ES 9 I 3.67 3.34
422ES 9 II 3.02 422ES 11 I 2.55 1.98 422ES 11 II 1.92 422ES 11 III
1.47 422ES 12 I 3.76 7.65 422ES 12 II 9.80 422ES 12 III 9.39 422ES
13 I 5.79 5.31 422ES 13 II 5.04 422ES 13 III 5.11 422ES 14 I 4.08
3.50 422ES 14 II 2.93 422ES 16 I 2.62 3.60 422ES 16 II 2.72 422ES
16 III 5.45 422ES 17 I 7.25 7.57 422ES 17 II 7.89 422ES 18 I n.d.
2.30 422ES 18 II 2.56 422ES 18 III 2.04 422ES wt 9 I 0.02 0.02
422ES wt 9 II 0.01 422ES wt 9 III 0.00 422ES wt 9 IV 0.05 422ES wt
9 V n.d. 422ES wt 14 I 0.05 0.05 422ES wt 14 II n.d. 422ES wt 14
III n.d. Column 1 refers to the plant, independently originating
from the transformation, from which material was harvested (here,
"wt" refers to non-transformed plants). The extension of the names
of the plants by Latin numerals denotes different fruits of the
plant in question. Non-detectable amounts are marked "n.d.".
[0277] These results show that plants having a foreign nucleic acid
molecule coding for a protein having the activity of a bacterial
GFAT and coding for a protein having the activity of a UDP-glucose
dehydrogenase have a considerably higher content of N-acetylated
glucosamine derivatives than correspondingly non-transformed
wild-type plants. Compared to plants having a foreign nucleic acid
molecule coding for a protein having the activity of a GFAT-1 and
coding for a protein having the activity of a UDP-glucose
dehydrogenase, plants comprising a foreign nucleic acid molecule
coding for a protein having the activity of a GFAT-2 and coding for
a protein having the activity of a UDP-glucose dehydrogenase have
an even higher content of N-acetylated glucosamine derivatives.
This is true both for leaf material and for fruits of the plants in
question.
h) Analysis of N-Acetylated Glucosamine Derivatives of Line 422 ES
by Mass Spectroscopy
[0278] Extracts of individual different fruits of the plant with
the name 422 ES 13 were examined by mass spectroscopy according to
the method described under General Methods Item 9 for the presence
of N-acetylated glucosamine derivatives. The following results were
obtained:
TABLE-US-00012 TABLE 8 Detection of the metabolites glucosamine
(GlcN), N-acetylglucosamine (GlcNAc), glucosamine phosphate
(GlcN-P), N-acetylglucosamine phosphate (GlcNAc-P) and
UDP-N-acetylglucosamine (UDP-GlcNAc) in fruits of the plant 422 ES
13 by mass spectroscopy. What is shown is the proportion of the
signal intensity (peak area) obtained for the stated metabolite in
the mass spectrum, based on the signal intensity for hexoses (m/z =
179) obtained in the same measurement, in percent. The different
measurements were carried out at the stated detector settings with
respect to sensitivity ("d.v.") and signal intensity ("cps")
(column 1). Column 2 denotes the plant, independently originating
from the transformation, from which material was harvested (here,
"wt" refers to non-transformed plants). The extension of the names
of the plants by Latin numerals denote different fruits of the
plant in question. Mass (m/z) with associated metabolites 178 220
258 300 302.5 Detector Sample GlcN GlcNAc GlcN-P GlcNAc-P
UDP-GlcNAc cps: 9.5-10 e4 422 ES 13 I 0.08 5.08 0.56 1.44 0.14
(d.v.: 2000) 422 ES 13 II 0.09 6.41 0.61 1.48 0.14 422 ES 13 III
0.09 5.95 1.05 1.51 0.18 wt 0.06 0.05 0.10 0.06 0.00 cps: 1.6-1.8
e5 422 ES 13 I 0.37 10.69 1.91 4.31 0.42 (d.v.: 2050) 422 ES 13 II
0.37 13.92 2.10 4.49 0.45 422 ES 13 III 0.30 12.96 3.43 3.98 0.55
wt 0.30 0.25 0.49 0.38 0.03 cps: 2.0-2.2 e5 422 ES 13 I 0.71 18.77
3.95 8.70 0.73 (d.v.: 2100) 422 ES 13 II 0.68 21.81 3.88 8.32 0.67
422 ES 13 III 0.48 19.82 6.25 7.14 0.84 wt 0.55 0.53 1.05 0.94
0.05
[0279] In parallel, via MS-MS measurements of samples 422 ES 13 I
and fruits of a wild-type plant (wt) using reference substances
(glucosamine, N-acetylgucosamine, glucosamine 6-phosphate,
glucosamine 1-phosphate, N-acetylgucosamine 6-phosphate,
N-acetylglucosamine 1-phosphate, UDP-N-acetylglucosamine) it was
analyzed whether the detected signal intensities (peak areas) in
question of the MS spectra were really due to the presence of the
corresponding metabolite or the corresponding isomeric metabolites
of the same mass, or whether the signal intensities in question in
the MS spectrum were possibly caused by interference by signals of
other substances. The following observations were made:
[0280] Glucosamine (GlcN, m/z=178): The highest amounts of GlcN
detected in the MS spectra of samples 422 ES 13 I and wt were in
the range of the lower detection limit. In the MS spectrum, no
significant differences between the sample 422 ES 13 I and the wt
samples were noticed. Accordingly, it was not possible to determine
with any degree of certainty whether the samples contained
GlcN.
[0281] N-Acetylglucosamine (GlcNAc, m/z=220): The most significant
differences in the MS spectra of samples 422 ES 13 and the wt
sample were found for this metabolite. In the MS spectra of samples
422 ES 13 I, 422 ES 13 II and 422 ES 13 III, considerable amounts
of GlcNAc were detected. The corresponding MS-MS spectrum for the
sample 422 ES 13 I corresponds to the spectrum of the reference
substance (N-acetylglucosamine) and has, if any, only very small
amounts of substances which may interfere with the relevant signal
in the MS spectrum. In contrast, in the MS spectrum of the wt
sample the signal intensity for m/z=220 was very low. The MS-MS
spectrum of the wt sample showed that GlcNAc is only present in
traces, if at all. The MS-MS spectrum very clearly showed that the
signal intensity determined for m/z=220 of the wt sample in the MS
spectrum was the result mainly of other substances interfering with
the signal.
[0282] Glucosamine phosphates (GlcN-P, m/z=258): The signal
intensity of the MS spectra for the wt sample is considerably lower
than for samples 422 ES 13 I, 422 ES 13 II and 422 ES 13 III. All
samples measured by MS-MS show that the signal for m/z=258 is not
only due to the presence of GlcN-P but also to interference of the
signal by other substances. The MS-MS spectrum of the wt sample
showed that only traces of GlcN-P are present, if any. In contrast,
the corresponding signal for sample 422 ES 13 I in the MS-MS
spectrum showed the presence of significant amounts of GlcN-P in
the relevant signal of the MS spectrum.
[0283] N-Acetylglucosamine phosphate (GlcNAc-P, m/z=300): For the
wt sample, the signal intensities for m/z=300 in the MS spectrum
are substantially lower than for the samples 422 ES 13 I, 422 ES 13
II and 422 ES 13 III. The values determined by MS-MS for the wt
sample show that, if any, only traces of GlcNAc-P are present. In
contrast, for sample 422 ES 13 I it was possible to demonstrate by
MS-MS measurement that the predominant part of the signal intensity
determined for m/z=300 in the MS spectrum of this sample is due to
GlcNAc-P.
[0284] UDP-N-Acetylglucosamine (UDP-GlcNAc, m/z=302.5): In the
wild-type, the signal intensities of the MS spectrum are
considerably lower than in samples 422 ES 13 I, 422 ES II and 422
ES III. The corresponding MS-MS spectra show that in all samples a
certain part of the signal intensity of the MS spectra is not only
due to the presence of UDP-GlcNAc, but also due to signal
interference by other substances. However, the MS-MS measurements
showed that compared to the signal-interfering substances, the
proportion of UDP-GlcNAc in the MS spectra of sample 422 ES 13 I is
substantially higher than for the wt sample.
19. Production of Plants which Synthesize Glucosaminoglycans
[0285] To determine whether plants having an increased content of
N-acetylated glucosamine derivatives are suitable for producing
plants having an increased glucosaminoglycan content, at first
plants expressing a glucosaminoglycan synthase (hyaluronan
synthase) were generated.
a) Plants Comprising a Nucleic Acid Molecule Coding for a Protein
Having the Activity of a Hyaluronan Synthase
[0286] Potato plants (cultivar Desiree) and tomato plants (cultivar
Moneymaker) were transformed using the method given under General
Methods Item 1 (potato plants) and under General Methods Item 2
(tomato plants) respectively, with the plant expression vector IC
341-222 which comprises a coding nucleic acid sequence for a
protein having the activity of a hyaluronan synthase from
Paramecium bursaria Chlorella Virus 1 under the control of the
promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa
et al., 1989, EMBO J. 8, 23-29). The transgenic lines obtained,
which are transformed with the plasmid IC 341-222, were named 365
ES (potato plants) and 367 ES (tomato plants), respectively.
b) Analysis of the Lines 365 ES
[0287] Individual plants of the line 365 ES were cultivated in soil
in 6 cm pots in a greenhouse. In each case about 0.3 g of material
of potato tubers of the individual plants was worked up using the
method described under General Methods Item 5. The amount of the
hyaluronan present in the respective plant extracts was determined
using the method described under General Methods Item 7. Here, the
supernatant obtained after centrifugation was diluted 1:10 to
determine the hyaluronan content. The following results were
obtained for selected plants:
TABLE-US-00013 TABLE 9 Amount of hyaluronan (in .mu.g per gram of
fresh weight) produced by independent selected transgenic plants of
line 365 ES. Plant Hyaluronan [.mu.g/g FW] 365 ES 13 47 365 ES 74
68 wt n.d. Column 1 refers to the plant from which tuber material
was harvested (here, "wt" refers to non-transformed plants). Column
2 states the value for the amount of hyaluronan determined in
leaves of the plant in question. Non-detectable amounts are marked
"n.d.".
c) Analysis of Plants of Line 367 ES
[0288] From different selected tomato plants of line 367 ES which
had been cultivated in soil in a greenhouse, in each case 1 leaf
was harvested and frozen in liquid nitrogen. Further work-up and
determination of the hyaluronan content was carried out as
described in Example 19b) for tubers of potato plants. The
following results were obtained:
TABLE-US-00014 TABLE 10 Amount of hyaluronan (in .mu.g per gram of
fresh weight) produced in leaves of independent selected transgenic
plants of lines 367 ES. Plant Hyaluronan [.mu.g/g FW] 367 ES 25
57.19 367 ES 42 88.99 wt 0.06 Column 1 refers to the plant from
which leaf material was harvested (here, "wt" refers to
non-transformed plants). Column 2 states the value of the amount of
hyaluronan determined in leaves of the plants in question.
20. Plants Comprising a Foreign Nucleic Acid Molecule Coding for a
Protein Having The Activity of a UDP-Glucose Dehydrogenase and a
Nucleic Acid Molecule Coding for a Protein Having the Activity of a
Glucosaminoglycan Synthase
[0289] Some glucosaminoglycan synthases (such as, for example,
hyaluronan synthase) require, as substrate, N-acetylated
glucosamine derivatives and UDP-GlcA. Accordingly, we first
generated plants having an increased activity of a protein having
the activity of a UDP-glucose dehydrogenase and an increased
activity of a protein having the activity of a hyaluronan
synthase.
a) Production of Potato Plants
[0290] Potato plants of line 365 ES 74 (see Example 19b)) were
transformed using the method given under General Methods Item 1
with the plant expression vector IC 349-222 comprising a coding
nucleic acid sequence for a protein having the activity of a
UDP-glucose dehydrogenase under the control of the 35S promoter.
The transgenic lines obtained, which are transformed with the
plasmid IC 349-222, were named 423 ES.
b) Analysis of Plants of Line 423 ES
[0291] Plants of line 423 ES were cultivated in soil in 6 cm pots
in a greenhouse. In each case about 0.3 g to 0.8 g of leaf
material, harvested from individual plants, was worked up using the
method described under General Methods Item 5, and the content of
Hyaluronan was determined using the method described under General
Methods Item 7. For individual plants having an increased content
of N-acetylglucosamine derivatives, the following results were
obtained:
TABLE-US-00015 TABLE 11 Amount of hyaluronan (in .mu.g per gram of
fresh weight) measured in leaves of independent transgenic plants
of line 423 ES. Plant Hyaluronan [.mu.g/g FW] 423ES 1 328.75 423ES
3 210.38 423ES 5 340.99 423ES 6 250.88 423ES 7 214.53 423ES 8
309.22 423ES 9 253.31 423ES 10 229.61 423ES 11 234.40 423ES 12
480.22 423ES 13 253.63 423ES 14 221.77 423ES 15 202.46 423ES 17
281.46 423ES 18 310.41 423ES 19 268.91 423ES 20 394.04 423ES 21
462.64 423ES 24 438.33 423ES 25 419.50 423ES 26 342.89 423ES 27
383.32 423ES 28 236.83 423ES 29 332.63 423ES 32 254.88 423ES 33
283.31 423ES 35 276.60 423ES 36 308.85 423ES 38 307.72 423ES 41
259.89 423ES 43 244.62 423ES 47 229.25 423ES 48 238.22 423ES 49
285.19 423ES 51 213.97 423ES 53 328.76 423ES 54 358.23 423ES 55
154.06 423ES 59 276.32 423ES 60 498.70 423ES 61 300.97 423ES 62
292.08 423ES 65 230.38 423ES 67 267.54 423ES 68 370.08 wt 1 0.38 wt
2 0.12 wt 3 0.07 wt 4 n.d. wt 5 0.47 wt 6 n.d. wt 7 0.05 wt 8 0.05
wt 9 0.10 wt 10 n.d. 365ES 74-1 348.43 365ES 74-2 214.59 365ES 74-3
391.88 365ES 74-4 442.60 365ES 74-5 293.01 365ES 74-6 323.47 365ES
74-7 464.21 365ES 74-8 341.32 365ES 74-9 338.93 365ES 74-10 438.55
Column 1 refers in each case to the plants, independently
originating from the transformation, from which material was
harvested (here, "wt 1" to "wt 10" refer to independent
non-transformed plants). For comparison, values for 10 different
progeny of plants of line 365 ES used as starting line for the
transformation (365 ES-1 to 365 ES-10) are shown. Non-detectable
amounts are marked "n.d.".
[0292] It can be seen from the results that plants comprising a
foreign nucleic acid molecule coding for a protein having the
activity of a UDP-glucose-dehydrogenase and a nucleic acid molecule
coding for a protein having the activity of a hyaluronan synthase
do not synthesize any statically significant increased amounts of
hyaluronan compared to plants having only a nucleic acid molecule
coding for a protein having the activity of a hyaluronan
synthase.
21. Plants Synthesizing Increased Amounts of Glucosaminoglycan
a) Production of Tomato Plants Synthesizing Increased Amounts of
Glucosaminoglycan
[0293] Tomato plants of lines 367 ES 25 (see example 19c)), having
a nucleic acid molecule coding for a hyaluronan synthase were
transformed again using the method given under General Methods Item
2 with the plant expression vectors IC 372-256 or IC 375-271
comprising nucleic acid molecules coding for different isoforms of
proteins having the activity of a GFAT.
[0294] The transgenic tomato plants obtained after transformation
of line 367 ES 25, with the plasmid IC 372-256 (GFAT-2), were named
399 ES.
[0295] The transgenic tomato plants obtained after transformation
of line 367 ES 25 with the plasmid IC 375-271 (bacterial GFAT),
were named 405 ES.
b) Analysis of Lines 399 ES and 405 ES
[0296] Ripe fruits were harvested from different tomato plants of
lines 399 ES and 405 ES cultivated in soil in a greenhouse, and the
hyaluronan content was determined as described under General
Methods Item 7. The following results were obtained:
TABLE-US-00016 TABLE 12 Amount of hyaluronan ("HA" in .mu.g per
gram of fresh weight) measured in fruits of independent transgenic
plants of lines 399 ES and 405 ES. HA Mean Sample [.mu.g/g FW]
[.mu.g/g FW] 399ES 1 I 63.38 87.02 399ES 1 II 96.45 399ES 1 III
101.23 399ES 11 I 388.83 292.79 399ES 11 II 244.01 399ES 11 III
254.91 399ES 11 IV 285.72 399ES 11 V 297.99 399ES 11 VI 285.29 wt I
0.02 0.01 wt II 0.02 wt III 0.01 wt IV n.d. 367ES 25-1 I 9.77 12.04
367ES 25-1 II 8.21 367ES 25-1 III 18.04 367ES 25-1 IV 13.86 367ES
25-1 V 10.33 367ES 25-2 I 9.31 11.96 367ES 25-2 II 10.55 367ES 25-2
III 11.53 367ES 25-2 IV 16.54 367ES 25-2 V 11.86 367ES 25-3 I 6.99
8.51 367ES 25-3 II 7.94 367ES 25-3 III 9.23 367ES 25-3 IV 7.09
367ES 25-3 V 11.28 405ES 5 I 207.20 254.94 405ES 5 II 302.67 405ES
10 I 1232.38 1074.94 405ES 10 II 917.50 wt I 0.86 0.46 wt II 0.06
367ES 25-8 I 136.67 155.70 367ES 25-8 II 174.72 367ES 25-9 I 37.76
Column 1 refers to the plants, independently originating from the
transformation, from which material was harvested (here, "wt"
refers to non-transformed plants). For comparison, values of
different progeny of plants of line 367 ES used as starting line
for the transformation are shown. The extensions of the names of
the plants by Latin numerals denote different fruits of the plant
in question. Non-detectable amounts are marked "n.d.".
[0297] These results show that plants comprising foreign nucleic
acid molecules coding for a glucosaminoglycan synthase and coding
for a protein having the activity of a UDP-glucose dehydrogenase
and coding for a protein having the activity of a GFAT-2 or a
bacterial GFAT synthesize considerably higher amounts of
glucosaminoglycans than plants having only a foreign nucleic acid
molecule coding for a glucosaminoglycan synthase.
c) Production of Potato Plants Synthesizing Increased Amounts of
Glucosaminoglycan
[0298] Potato plants of lines 365 ES 74 (see example 19b))
comprising a nucleic acid molecule coding for a hyaluronan synthase
were transformed again using the method stated under General
Methods Item 1 with the plant expression vectors IC 376-271, IC
372-256 or IC 375-271 comprising nucleic acid molecules coding for
different isoforms of proteins having the activity of a GFAT.
[0299] The transgenic potato plants obtained after transformation
of line 365 ES 74 with the plasmid IC 376-271 (GFAT-1), were named
409 ES.
[0300] The transgenic potato plants obtained after transformation
of line 365 ES 74 with the plasmid IC 372-256 (GFAT-2), were named
396 ES.
[0301] The transgenic potato plants obtained after transformation
of line 365 ES 74 with the plasmid IC 375-271 (bacterial GFAT),
were named 404 ES.
d) Analysis of Lines 396 ES, 404 ES and 409 ES
[0302] Leaf and/or tuber material was harvested from different
potato plants of lines 396 ES (GFAT-2), 404 ES (bacterial GFAT) and
409 ES (GFAT-1) cultivated in soil in a greenhouse, and the
hyaluronan content was determined as described under General
Methods Item 7. The following results were obtained for plants of
line 409 ES:
TABLE-US-00017 TABLE 13 Amount of hyaluronan ("HA" in .mu.g per
gram of fresh weight) measured in leaves and tubers of independent
transgenic plants of line 409 ES. HA in HA in leaves tubers Plant
[.mu.g/g FW] [.mu.g/g FW] 409 ES 2 54.01 409 ES 3 68.75 212.24 409
ES 4 59.80 111.54 409 ES 5 26.90 409 ES 6 38.01 182.39 409 ES 7
25.80 95.68 409 ES 8 51.92 99.35 409 ES 9 48.43 168.61 409 ES 10
52.52 409 ES 13 55.87 409 ES 14 45.91 143.96 409 ES 15 52.76 409 ES
16 60.28 409 ES 22 69.47 114.97 409 ES 23 108.67 409 ES 26 38.81
409 ES 27 24.71 126.74 409 ES 28 66.95 409 ES 29 79.58 164.66 wt-1
n.d. wt-2 n.d. wt-3 n.d. wt-4 n.d. 365 ES 74-1 25.19 365 ES 74-2
31.15 365 ES 74-3 72.96 365 ES 74-4 35.98 365 ES 74-5 40.18 123.66
365 ES 74-6 37.70 Column 1 refers to the plants, independently
originating from the transformation, from which material was
harvested (here, "wt" refers to non-transformed plants). Values for
different progeny of plants of line 365 ES 74, which was used as
starting line for the transformation, are shown for comparison.
Non-detectable amounts are marked "n.d.".
[0303] The following results were obtained for plants of line 396
ES:
TABLE-US-00018 TABLE 14 Amount of hyaluronan ("HA" in .mu.g per
gram of fresh weight) measured in leaves and tubers of independent
transgenic plants of line 396 ES. HA in HA in leaves tubers Plant
[.mu.g/g FW] [.mu.g/g FW] 396 ES 2 470.93 396 ES 9 735.40 396 ES 11
938.33 396 ES 15 393.64 396 ES 16 416.43 396 ES 17 426.79 396 ES 23
271.85 396 ES 24 443.57 396 ES 25 801.58 396 ES 28 484.76 396 ES 30
224.06 396 ES 32 941.89 396 ES 33 1295.98 396 ES 34 796.79 396 ES
36 204.49 396 ES 36 860.54 396 ES 42 1445.51 396 ES 44 1312.56 396
ES 48 461.05 396 ES 49 538.75 396 ES 50 619.23 396 ES 51 1160.57
396 ES 57 428.33 396 ES 57 807.97 365 ES 74-1 265.10 366 ES 74-2
91.84 365 ES 74-3 193.50 367 ES 74-4 175.48 365 ES 74-5 73.90 368
ES 74-6 168.68 365 ES 74-7 67.58 369 ES 74-8 121.89 365 ES 74-9
62.23 370 ES 74-10 275.24 365 ES 74-11 134.56 wt-1 0.07 2.27 wt-2
0.11 wt-3 0.12 1.07 wt-4 0.04 0.78 wt-5 0.10 wt-5 0.24 Column 1
refers to the plants, independently originating from the
transformation, from which material was harvested (here, "wt"
refers to non-transformed plants). Values for different progeny of
plants of line 365 ES 74, which was used as starting line for the
transformation, are shown for comparison.
[0304] The following results were obtained for plants of 404
ES:
TABLE-US-00019 TABLE 15 Amount of hyaluronan (in .mu.g per gram of
fresh weight) measured in leaves of independent transgenic plants
of line 404 ES. Plant Hyaluronan in leaves [.mu.g/g FW] 404 ES 1
801.14 404 ES 6 365.15 404 ES 7 218.42 404 ES 8 521.92 404 ES 9
366.46 404 ES 10 226.83 404 ES 11 231.39 404 ES 13 1547.12 404 ES
14 616.79 404 ES 15 832.32 404 ES 20 581.11 404 ES 21 489.73 404 ES
23 817.91 404 ES 24 434.06 404 ES 26 205.00 404 ES 28 359.96 404 ES
29 1146.68 404 ES 34 310.76 404 ES 35 1388.51 404 ES 36 1095.11 404
ES 37 533.89 404 ES 38 651.12 404 ES 39 353.74 404 ES 40 371.88 404
ES 42 849.43 404 ES 43 479.34 404 ES 44 921.11 404 ES 46 846.81 404
ES 48 302.54 wt-1 0.20 wt-2 0.30 wt-3 0.19 wt-4 0.39 wt-5 0.20 365
ES 74-1 72.44 365 ES 74-2 135.60 365 ES 74-3 19.56 365 ES 74-4
114.83 365 ES 74-5 73.77 Column 1 refers to the plants,
independently originating from the transformation, from which
material was harvested here, ("wt" refers to non-transformed
plants). Values for different progeny of plants of line 365 ES 74,
which was used as starting line for the transformation, are shown
for comparison.
[0305] These results show that plants comprising foreign nucleic
acid molecules coding for a glucosaminoglycan synthase and coding
for a protein having the activity of a UDP-glucose dehydrogenase
and coding for a protein having the activity of a GFAT-2 or a
bacterial GFAT synthesize considerably higher amounts of
glucosaminoglycan than plants comprising foreign nucleic acid
molecules coding for a glucosaminoglycan synthase and coding for a
protein having the activity of a UDP-glucose dehydrogenase and
coding for a protein having the activity of a GFAT-1.
e) Production of Plants Comprising Foreign Nucleic Acid Molecules
Coding for a Hyaluronan Synthase and a Protein Having the Activity
of a Bacterial GFAT
[0306] Potato plants of line 365 ES 74 (see example 19b)),
comprising a nucleic acid molecule coding for a hyaluronan synthase
were transformed again using the method given under General Methods
Item 1 with the plant expression vector IC 398-311 comprising
nucleic acid molecules coding for a protein having the activity of
a bacterial GFAT. The lines originating from this transformation
were named 433 ES.
f) Analysis of Line 433 ES
[0307] Leaf and/or tuber material was harvested from different
potato plants of line 433 ES cultivated in soil in a greenhouse,
and the hyaluronan content was determined as described under
General Methods Item 7. The following results were obtained for
plants of line 433 ES:
TABLE-US-00020 TABLE 16 Amount of hyaluronan ("HA" in .mu.g per
gram of fresh weight) measured in leaves and tubers of independent
transgenic plants of line 433 ES. HA in HA in tubers leaves
[.mu.g/g Plant [.mu.g/g FW] FW] 433ES 1 111.84 126.70 433ES 3
303.34 203.16 433ES 4 3142.41 433ES 5 312.98 825.96 433ES 7 1492.94
433ES 8 914.03 433ES 9 1858.68 433ES 10 357.90 433ES 11 5962.82
433ES 12 662.99 433ES 13 626.52 624.33 433ES 14 665.23 433ES 15
601.36 433ES 16 3416.94 433ES 18 781.02 433ES 19 3294.09 433ES 20
1348.85 975.18 433ES 21 937.92 433ES 22 1086.45 433ES 23 1327.28
433ES 24 340.80 76.00 433ES 25 1529.95 433ES 26 375.53 433ES 27
425.65 433ES 28 1850.99 294.98 433ES 30 2512.40 433ES 31 3337.54
433ES 32 1583.60 433ES 34 3552.44 433ES 35 5419.43 433ES 36 902.01
433ES 37 829.35 433ES 38 1536.55 wt-1 0.40 n.d. wt-2 0.34 n.d. wt-3
n.d. 365 ES 74-1 265.1 366 ES 74-2 91.84 365 ES 74-3 193.5 367 ES
74-4 175.48 365 ES 74-5 73.9 368 ES 74-6 168.68 365 ES 74-7 67.58
369 ES 74-8 121.89 365 ES 74-9 62.23 370 ES 74-10 275.24 365 ES
74-11 134.56 Column 1 refers to the plants, independently
originating from the transformation, from which material was
harvested (here, "wt" refers to non-transformed plants). Values for
different progeny of plants of line 365 ES 74, which was used as
starting line for the transformation, are shown for comparison. The
values for line 365 ES 74 correspond to those in Table 14, since
all plants were cultivated simultaneously in a greenhouse.
[0308] These results show that plants comprising foreign nucleic
acid molecules coding for a glucosaminoglycan synthase and coding
for a protein having the activity of a bacterial GFAT synthesize
considerably higher amounts of glucosaminoglycan than plants having
only foreign nucleic acid molecules coding for a glucosaminoglycan
synthase.
22. Summary of the Results
[0309] The results in Example 16 show that plants comprising a
nucleic acid molecule coding for a protein having the activity of a
bacterial GFAT have considerably increased contents of N-acetylated
glucosamine derivatives compared to non-transformed wild-type
plants.
[0310] The results in Example 17 show that plants comprising a
nucleic acid molecule coding for a protein having the activity of a
GFAT-2 have considerably higher contents of N-acetylated
glucosamine derivatives than non-transformed wild-type plants.
[0311] All transformed plants described in Example 18 have, in
addition to nucleic acid molecules coding for different isoforms of
a protein having the activity of a GFAT, in each case the same
nucleic acid molecule coding for a protein having the activity of a
UDP-glucose dehydrogenase. Accordingly, the essential difference of
the transformed plants described in Example 18 are the different
foreign nucleic acid molecules coding for the different isoforms of
a protein having the activity of a GFAT. Example 18b) shows that
the content of N-acetylated glucosamine derivatives in plants
having a foreign nucleic acid molecule coding for a protein having
the activity of a GFAT-1 is increased only slightly compared to
non-transformed plants.
[0312] Furthermore, it can be seen from Example 18d) that plants
comprising a foreign nucleic acid molecule coding for a protein
having the activity of a GFAT-2 have a considerably higher content
of N-acetylated glucosamine derivatives than non-transformed
wild-type plants. The content of N-acetylated glucosamine
derivatives in plants having a foreign nucleic acid molecule coding
for a protein having the activity of a GFAT-2 is also considerably
higher than in plants having a foreign nucleic acid molecule coding
for a protein having the activity of a GFAT-1.
[0313] Furthermore, it can be seen from Examples 18f) and g) that
plants comprising a foreign nucleic acid molecule coding for a
protein having the activity of a bacterial GFAT have even higher
contents of N-acetylated glucosamine derivatives than plants
comprising a foreign nucleic acid molecule coding for a protein
having the activity of a GFAT-2.
[0314] The results in Example 21f) show that plants comprising
foreign nucleic acid molecules coding for a glucosaminoglycan
synthase and coding for a protein having the activity of a
bacterial GFAT synthesize considerably higher amounts of
glucosaminoglycan than plants having only foreign nucleic acid
molecules coding for a glucosaminoglycan synthase.
[0315] Thus, it can be concluded that the amount of
glucosaminoglycans synthesized in plants can be increased
considerably by generating plants which, in addition to foreign
nucleic acid molecules coding for a glucosaminoglycan synthase,
additionally comprise foreign nucleic acid molecules coding for a
protein having the activity of a bacterial GFAT.
[0316] All transformed plants whose results are shown in Examples
21b) and d) have, in addition to nucleic acid molecules coding for
different isoforms of a protein having the activity of a GFAT, also
foreign nucleic acid molecules coding for a protein having the
activity of a UDP-glucose dehydrogenase and foreign nucleic acid
molecules coding for a glucosaminoglycan synthase. The essential
difference between the transformed plants whose results are shown
in Examples 21b) and d) accordingly consists in the different
nucleic acid molecules coding for the different isoforms of a
protein having the activity of a GFAT.
[0317] The results shown in Example 21b) show that the content of
glucosaminoglycans in plants having a foreign nucleic acid molecule
coding for a protein having the activity of a GFAT-2 or coding for
a protein having the activity of a bacterial GFAT is increased
considerably compared to plants having only the activity of a
glucosaminoglycan synthase.
[0318] The results shown in Example 21d) show that plants
comprising a foreign nucleic acid molecule coding a protein having
the activity of a GFAT-1 contain a slightly higher amount of
glucosaminoglycans than plants having only the activity of a
glucosaminoglycan synthase. In contrast, the content of
glucosaminoglycans in plants comprising a foreign nucleic acid
molecule coding for a protein having the activity of a GFAT-2 is
considerably higher than in plants having a foreign nucleic acid
molecule coding for a protein having the activity of a GFAT-1.
Furthermore, it can be seen from Example 21d) that individual
plants comprising a foreign nucleic acid molecule coding for a
protein having the activity of a bacterial GFAT contain even higher
amounts of glucosaminoglycans than plants having a foreign nucleic
acid molecule coding for a protein having the activity of a
GFAT-2.
[0319] The results in Example 20b) show that plants comprising a
foreign nucleic acid molecule coding for a protein having the
activity of a UDP-glucose dehydrogenase and a nucleic acid molecule
coding for a protein having the activity of a glucosaminoglycan
synthase do not have any statistically significantly increased
amounts of glucosaminoglycan compared to plants comprising only a
foreign nucleic acid molecule coding for a protein having the
activity of a glucosaminoglycan synthase.
[0320] To conclude, the results shown indicate that the
considerable increases in the amounts of glucosaminoglycans in
plants comprising foreign nucleic acid molecules coding for a
protein having the activity of a UDP-glucose dehydrogenase and
coding for a protein having the activity of a glucosaminoglycan
synthase and having the activity of a GFAT-2 or having the activity
of a bacterial GFAT is not due to the presence of the foreign
nucleic acid molecules having the activity of a UDP-glucose
dehydrogenase but to the presence of nucleic acid molecules having
the activity of a GFAT-2 or having the activity of a bacterial
GFAT.
[0321] Since hyaluronan synthases used in an exemplary manner as
proteins having the activity of a glucosaminoglycan synthase
require, as substrates, both UDP-Glc-NAc and UDP-GlcA, it may also
be concluded from the results shown that the increased amounts of
hyaluronan (glucosaminoglycan) are due to increased amounts of
N-acetylated glucosamine derivatives and not to increased amounts
of UDP-GlcA in these plants.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 1707
<212> TYPE: DNA <213> ORGANISM: Paramecium bursaria
Chlorella Virus 1 <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)..(1707) <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: PB42580
<309> DATABASE ENTRY DATE: 1995-12-24 <313> RELEVANT
RESIDUES IN SEQ ID NO: (50903)..(52609) <400> SEQUENCE: 1 atg
ggt aaa aat ata atc ata atg gtt tcg tgg tac acc atc ata act 48 Met
Gly Lys Asn Ile Ile Ile Met Val Ser Trp Tyr Thr Ile Ile Thr 1 5 10
15 tca aat cta atc gcg gtt gga gga gcc tct cta atc ttg gct ccg gca
96 Ser Asn Leu Ile Ala Val Gly Gly Ala Ser Leu Ile Leu Ala Pro Ala
20 25 30 att act ggg tat gtt cta cat tgg aat att gct ctc tcg aca
atc tgg 144 Ile Thr Gly Tyr Val Leu His Trp Asn Ile Ala Leu Ser Thr
Ile Trp 35 40 45 gga gta tca gct tat ggt att ttc gtt ttt ggg ttt
ttc ctt gca caa 192 Gly Val Ser Ala Tyr Gly Ile Phe Val Phe Gly Phe
Phe Leu Ala Gln 50 55 60 gtt tta ttt tca gaa ctg aac agg aaa cgt
ctt cgc aag tgg att tct 240 Val Leu Phe Ser Glu Leu Asn Arg Lys Arg
Leu Arg Lys Trp Ile Ser 65 70 75 80 ctc aga cct aag ggt tgg aat gat
gtt cgt ttg gct gtg atc att gct 288 Leu Arg Pro Lys Gly Trp Asn Asp
Val Arg Leu Ala Val Ile Ile Ala 85 90 95 gga tat cgc gag gat cct
tat atg ttc cag aag tgc ctc gag tct gta 336 Gly Tyr Arg Glu Asp Pro
Tyr Met Phe Gln Lys Cys Leu Glu Ser Val 100 105 110 cgt gac tct gat
tat ggc aac gtt gcc cgt ctg att tgt gtg att gac 384 Arg Asp Ser Asp
Tyr Gly Asn Val Ala Arg Leu Ile Cys Val Ile Asp 115 120 125 ggt gat
gag gac gat gat atg agg atg gct gcc gtt tac aag gcg atc 432 Gly Asp
Glu Asp Asp Asp Met Arg Met Ala Ala Val Tyr Lys Ala Ile 130 135 140
tac aat gat aat atc aag aag ccc gag ttt gtt ctg tgt gag tca gac 480
Tyr Asn Asp Asn Ile Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp 145
150 155 160 gac aag gaa ggt gaa cgc atc gac tct gat ttc tct cgc gac
att tgt 528 Asp Lys Glu Gly Glu Arg Ile Asp Ser Asp Phe Ser Arg Asp
Ile Cys 165 170 175 gtc ctc cag cct cat cgt gga aaa cgg gag tgt ctt
tat act ggg ttt 576 Val Leu Gln Pro His Arg Gly Lys Arg Glu Cys Leu
Tyr Thr Gly Phe 180 185 190 caa ctt gca aag atg gac ccc agt gtc aat
gct gtc gtt ctg att gac 624 Gln Leu Ala Lys Met Asp Pro Ser Val Asn
Ala Val Val Leu Ile Asp 195 200 205 agc gat acc gtt ctc gag aag gat
gct att ctg gaa gtt gta tac cca 672 Ser Asp Thr Val Leu Glu Lys Asp
Ala Ile Leu Glu Val Val Tyr Pro 210 215 220 ctt gca tgc gat ccc gag
atc caa gcc gtt gca ggt gag tgt aag att 720 Leu Ala Cys Asp Pro Glu
Ile Gln Ala Val Ala Gly Glu Cys Lys Ile 225 230 235 240 tgg aac aca
gac act ctt ttg agt ctt ctc gtc gct tgg cgg tac tat 768 Trp Asn Thr
Asp Thr Leu Leu Ser Leu Leu Val Ala Trp Arg Tyr Tyr 245 250 255 tct
gcg ttt tgt gtg gag agg agt gcc cag tct ttt ttc agg act gtt 816 Ser
Ala Phe Cys Val Glu Arg Ser Ala Gln Ser Phe Phe Arg Thr Val 260 265
270 cag tgc gtt ggg ggg cca ctg ggt gcc tac aag att gat atc att aag
864 Gln Cys Val Gly Gly Pro Leu Gly Ala Tyr Lys Ile Asp Ile Ile Lys
275 280 285 gag att aag gac ccc tgg att tcc cag cgc ttt ctt ggt cag
aag tgt 912 Glu Ile Lys Asp Pro Trp Ile Ser Gln Arg Phe Leu Gly Gln
Lys Cys 290 295 300 act tac ggt gac gac cgc cgg cta acc aac gag atc
ttg atg cgt ggt 960 Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile
Leu Met Arg Gly 305 310 315 320 aaa aag gtt gtg ttc act cca ttt gct
gtt ggt tgg tct gac agt ccg 1008 Lys Lys Val Val Phe Thr Pro Phe
Ala Val Gly Trp Ser Asp Ser Pro 325 330 335 acc aat gtg ttt cgg tac
atc gtt cag cag acc cgc tgg agt aag tcg 1056 Thr Asn Val Phe Arg
Tyr Ile Val Gln Gln Thr Arg Trp Ser Lys Ser 340 345 350 tgg tgc cgc
gaa att tgg tac acc ctc ttc gcc gcg tgg aag cac ggt 1104 Trp Cys
Arg Glu Ile Trp Tyr Thr Leu Phe Ala Ala Trp Lys His Gly 355 360 365
ttg tct gga att tgg ctg gcc ttt gaa tgt ttg tat caa att aca tac
1152 Leu Ser Gly Ile Trp Leu Ala Phe Glu Cys Leu Tyr Gln Ile Thr
Tyr 370 375 380 ttc ttc ctc gtg att tac ctc ttt tct cgc cta gcc gtt
gag gcc gac 1200 Phe Phe Leu Val Ile Tyr Leu Phe Ser Arg Leu Ala
Val Glu Ala Asp 385 390 395 400 cct cgc gcc cag aca gcc acg gtg att
gtg agc acc acg gtt gca ttg 1248 Pro Arg Ala Gln Thr Ala Thr Val
Ile Val Ser Thr Thr Val Ala Leu 405 410 415 att aag tgt ggg tat ttt
tca ttc cga gcc aag gat att cgg gcg ttt 1296 Ile Lys Cys Gly Tyr
Phe Ser Phe Arg Ala Lys Asp Ile Arg Ala Phe 420 425 430 tac ttt gtg
ctt tat aca ttt gtt tac ttt ttc tgt atg att ccg gcc 1344 Tyr Phe
Val Leu Tyr Thr Phe Val Tyr Phe Phe Cys Met Ile Pro Ala 435 440 445
agg att act gca atg atg acg ctt tgg gac att ggc tgg ggt act cgc
1392 Arg Ile Thr Ala Met Met Thr Leu Trp Asp Ile Gly Trp Gly Thr
Arg 450 455 460 ggt gga aac gag aag cct tcc gtt ggc acc cgg gtc gct
ctg tgg gca 1440 Gly Gly Asn Glu Lys Pro Ser Val Gly Thr Arg Val
Ala Leu Trp Ala 465 470 475 480 aag caa tat ctc att gca tat atg tgg
tgg gcc gcg gtt gtt ggc gct 1488 Lys Gln Tyr Leu Ile Ala Tyr Met
Trp Trp Ala Ala Val Val Gly Ala 485 490 495 gga gtt tac agc atc gtc
cat aac tgg atg ttc gat tgg aat tct ctt 1536 Gly Val Tyr Ser Ile
Val His Asn Trp Met Phe Asp Trp Asn Ser Leu 500 505 510 tct tat cgt
ttt gct ttg gtt ggt att tgt tct tac att gtt ttt att 1584 Ser Tyr
Arg Phe Ala Leu Val Gly Ile Cys Ser Tyr Ile Val Phe Ile 515 520 525
gtt att gtg ctg gtg gtt tat ttc acc ggc aaa att acg act tgg aat
1632 Val Ile Val Leu Val Val Tyr Phe Thr Gly Lys Ile Thr Thr Trp
Asn 530 535 540 ttc acg aag ctt cag aag gag cta atc gag gat cgc gtt
ctg tac gat 1680 Phe Thr Lys Leu Gln Lys Glu Leu Ile Glu Asp Arg
Val Leu Tyr Asp 545 550 555 560 gca act acc aat gct cag tct gtg tga
1707 Ala Thr Thr Asn Ala Gln Ser Val 565 <210> SEQ ID NO 2
<211> LENGTH: 568 <212> TYPE: PRT <213> ORGANISM:
Paramecium bursaria Chlorella Virus 1 <400> SEQUENCE: 2 Met
Gly Lys Asn Ile Ile Ile Met Val Ser Trp Tyr Thr Ile Ile Thr 1 5 10
15 Ser Asn Leu Ile Ala Val Gly Gly Ala Ser Leu Ile Leu Ala Pro Ala
20 25 30 Ile Thr Gly Tyr Val Leu His Trp Asn Ile Ala Leu Ser Thr
Ile Trp 35 40 45 Gly Val Ser Ala Tyr Gly Ile Phe Val Phe Gly Phe
Phe Leu Ala Gln 50 55 60 Val Leu Phe Ser Glu Leu Asn Arg Lys Arg
Leu Arg Lys Trp Ile Ser 65 70 75 80 Leu Arg Pro Lys Gly Trp Asn Asp
Val Arg Leu Ala Val Ile Ile Ala 85 90 95 Gly Tyr Arg Glu Asp Pro
Tyr Met Phe Gln Lys Cys Leu Glu Ser Val 100 105 110 Arg Asp Ser Asp
Tyr Gly Asn Val Ala Arg Leu Ile Cys Val Ile Asp 115 120 125 Gly Asp
Glu Asp Asp Asp Met Arg Met Ala Ala Val Tyr Lys Ala Ile 130 135 140
Tyr Asn Asp Asn Ile Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp 145
150 155 160 Asp Lys Glu Gly Glu Arg Ile Asp Ser Asp Phe Ser Arg Asp
Ile Cys 165 170 175 Val Leu Gln Pro His Arg Gly Lys Arg Glu Cys Leu
Tyr Thr Gly Phe 180 185 190 Gln Leu Ala Lys Met Asp Pro Ser Val Asn
Ala Val Val Leu Ile Asp 195 200 205 Ser Asp Thr Val Leu Glu Lys Asp
Ala Ile Leu Glu Val Val Tyr Pro 210 215 220 Leu Ala Cys Asp Pro Glu
Ile Gln Ala Val Ala Gly Glu Cys Lys Ile 225 230 235 240 Trp Asn Thr
Asp Thr Leu Leu Ser Leu Leu Val Ala Trp Arg Tyr Tyr 245 250 255 Ser
Ala Phe Cys Val Glu Arg Ser Ala Gln Ser Phe Phe Arg Thr Val 260 265
270 Gln Cys Val Gly Gly Pro Leu Gly Ala Tyr Lys Ile Asp Ile Ile Lys
275 280 285 Glu Ile Lys Asp Pro Trp Ile Ser Gln Arg Phe Leu Gly Gln
Lys Cys 290 295 300 Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile
Leu Met Arg Gly 305 310 315 320 Lys Lys Val Val Phe Thr Pro Phe Ala
Val Gly Trp Ser Asp Ser Pro 325 330 335 Thr Asn Val Phe Arg Tyr Ile
Val Gln Gln Thr Arg Trp Ser Lys Ser 340 345 350 Trp Cys Arg Glu Ile
Trp Tyr Thr Leu Phe Ala Ala Trp Lys His Gly 355 360 365 Leu Ser Gly
Ile Trp Leu Ala Phe Glu Cys Leu Tyr Gln Ile Thr Tyr 370 375 380 Phe
Phe Leu Val Ile Tyr Leu Phe Ser Arg Leu Ala Val Glu Ala Asp 385 390
395 400 Pro Arg Ala Gln Thr Ala Thr Val Ile Val Ser Thr Thr Val Ala
Leu 405 410 415 Ile Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys Asp Ile
Arg Ala Phe 420 425 430 Tyr Phe Val Leu Tyr Thr Phe Val Tyr Phe Phe
Cys Met Ile Pro Ala 435 440 445 Arg Ile Thr Ala Met Met Thr Leu Trp
Asp Ile Gly Trp Gly Thr Arg 450 455 460 Gly Gly Asn Glu Lys Pro Ser
Val Gly Thr Arg Val Ala Leu Trp Ala 465 470 475 480 Lys Gln Tyr Leu
Ile Ala Tyr Met Trp Trp Ala Ala Val Val Gly Ala 485 490 495 Gly Val
Tyr Ser Ile Val His Asn Trp Met Phe Asp Trp Asn Ser Leu 500 505 510
Ser Tyr Arg Phe Ala Leu Val Gly Ile Cys Ser Tyr Ile Val Phe Ile 515
520 525 Val Ile Val Leu Val Val Tyr Phe Thr Gly Lys Ile Thr Thr Trp
Asn 530 535 540 Phe Thr Lys Leu Gln Lys Glu Leu Ile Glu Asp Arg Val
Leu Tyr Asp 545 550 555 560 Ala Thr Thr Asn Ala Gln Ser Val 565
<210> SEQ ID NO 3 <211> LENGTH: 1707 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic sequence encoding
Paramecium bursaria Chlorella Virus Hyaluronansynthase protein
<400> SEQUENCE: 3 atgggtaaga acattatcat tatggtgtcc tggtacacaa
ttattacaag taatctcatc 60 gcagttggtg gtgcatctct tattctcgct
ccagctatca ctggatatgt tcttcactgg 120 aacatcgccc tctcaactat
ttggggagtt tccgcatatg gtatttttgt tttcgggttc 180 tttttggctc
aggttctgtt ctcagagctc aatcgtaaga gactcaggaa gtggattagc 240
cttagaccaa aggggtggaa tgacgttcgt ctcgctgtca ttatcgctgg ctaccgtgaa
300 gatccttaca tgtttcaaaa gtgcttggaa tcagttaggg atagtgatta
tggcaacgtc 360 gctagactga tctgtgtgat tgatggagat gaggacgacg
atatgaggat ggcagctgtt 420 tataaggcta tctataatga taacattaag
aagcctgaat ttgttctttg cgagtctgat 480 gacaaggaag gagaacggat
tgattcagat ttctcacgtg atatctgcgt tctccaacct 540 catcgtggga
agcgtgaatg tctttataca ggtttccaac tcgccaaaat ggacccatca 600
gtgaacgctg tggttcttat cgatagtgat actgtgctgg agaaagatgc tatcttggag
660 gttgtttacc ctcttgcctg tgatcctgaa attcaagctg tggctggaga
gtgcaagatc 720 tggaacacag atactcttct ttctctgctt gtcgcatgga
gatattactc cgcattctgt 780 gtggagagga gcgctcaatc ctttttccgt
accgttcaat gcgttggtgg tcctttggga 840 gcttacaaaa ttgatatcat
caaggagatt aaggacccat ggattagtca aaggtttctt 900 ggtcagaagt
gcacttatgg cgatgatcgt agattgacta acgaaatcct tatgaggggc 960
aagaaagtcg tttttactcc atttgctgtc ggatggtctg attcacctac aaatgttttc
1020 cgttatattg tgcaacaaac acgttggagt aagagctggt gtagggagat
ctggtacact 1080 ttgttcgctg cttggaagca cgggcttagc ggaatttggc
ttgcttttga atgcctttac 1140 cagattacat actttttctt ggtgatctat
ttgttttcac gtcttgccgt cgaggctgac 1200 cctagagcac agactgcaac
tgtgattgtt tctactacag tcgcacttat taagtgtggc 1260 tatttcagtt
ttagagcaaa agatattaga gccttctatt ttgttttgta cacatttgtt 1320
tatttctttt gcatgattcc agctcgtatt accgctatga tgaccttgtg ggacatcgga
1380 tggggaacta gaggtggtaa cgaaaagcct tctgtgggaa caagggtggc
cctttgggca 1440 aaacaatatc tcatcgccta catgtggtgg gccgctgtcg
ttggtgccgg agtgtactca 1500 atcgttcata actggatgtt tgactggaac
tctttgagct atcgtttcgc tcttgtgggt 1560 atttgttctt acattgtttt
catcgtgatt gtgctcgttg tgtatttcac tggtaaaatc 1620 acaacctgga
atttcactaa acttcaaaag gaattgattg aagacagggt tctgtatgat 1680
gctactacca acgcccagtc agtttaa 1707 <210> SEQ ID NO 4
<211> LENGTH: 2298 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (150)..(2192) <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: BC050762.1
<309> DATABASE ENTRY DATE: 2005-03-08 <313> RELEVANT
RESIDUES IN SEQ ID NO: (150)..(2195) <400> SEQUENCE: 4
gagagcgaag cgagcgctga gtcggactgt cgggtctgag ctgtcgcatc ccagagtcct
60 ctcattgcca ccaccccggc ccgagctcac cctcgcttct gaagctctcc
gcgcgcccga 120 cagctcagcc ctcgcccgtg accaacatc atg tgc ggt ata ttt
gct tat tta 173 Met Cys Gly Ile Phe Ala Tyr Leu 1 5 aat tac cat gtt
cct cga aca aga cga gaa atc ttg gag aca cta atc 221 Asn Tyr His Val
Pro Arg Thr Arg Arg Glu Ile Leu Glu Thr Leu Ile 10 15 20 aaa ggc
ctt cag aga ctg gaa tac aga gga tat gat tct gct ggt gtg 269 Lys Gly
Leu Gln Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala Gly Val 25 30 35 40
gga ctt gac gga ggc aat gac aaa gac tgg gaa gcc aac gcc tgc aaa 317
Gly Leu Asp Gly Gly Asn Asp Lys Asp Trp Glu Ala Asn Ala Cys Lys 45
50 55 atc cag ctc att aag aag aaa gga aaa gtt aag gca ctg gat gaa
gaa 365 Ile Gln Leu Ile Lys Lys Lys Gly Lys Val Lys Ala Leu Asp Glu
Glu 60 65 70 gtt cac aaa caa caa gat atg gac ttg gat ata gaa ttt
gat gtg cat 413 Val His Lys Gln Gln Asp Met Asp Leu Asp Ile Glu Phe
Asp Val His 75 80 85 ctt gga ata gct cat acc cgt tgg gcg aca cat
gga gaa ccc aat cct 461 Leu Gly Ile Ala His Thr Arg Trp Ala Thr His
Gly Glu Pro Asn Pro 90 95 100 gtc aat agt cac ccc cag cgc tct gat
aaa aat aat gaa ttc att gtt 509 Val Asn Ser His Pro Gln Arg Ser Asp
Lys Asn Asn Glu Phe Ile Val 105 110 115 120 att cat aat gga atc atc
acc aac tac aaa gac ttg aaa aag ttt ctg 557 Ile His Asn Gly Ile Ile
Thr Asn Tyr Lys Asp Leu Lys Lys Phe Leu 125 130 135 gaa agc aaa ggc
tat gac ttt gaa tct gaa aca gac aca gaa acc att 605 Glu Ser Lys Gly
Tyr Asp Phe Glu Ser Glu Thr Asp Thr Glu Thr Ile 140 145 150 gcc aag
ctc gtc aag tac atg tat gac aac tgg gag agc cag gac gtc 653 Ala Lys
Leu Val Lys Tyr Met Tyr Asp Asn Trp Glu Ser Gln Asp Val 155 160 165
agt ttt acc acc ttg gtg gag aga gtt atc caa caa ttg gaa ggc gcc 701
Ser Phe Thr Thr Leu Val Glu Arg Val Ile Gln Gln Leu Glu Gly Ala 170
175 180 ttt gct ctt gtg ttt aaa agt gtc cat ttt ccc ggg caa gca gtt
ggc 749 Phe Ala Leu Val Phe Lys Ser Val His Phe Pro Gly Gln Ala Val
Gly 185 190 195 200 aca agg cga ggt agc cct ctc ttg att ggt gtg cgg
agt gaa cat aag 797 Thr Arg Arg Gly Ser Pro Leu Leu Ile Gly Val Arg
Ser Glu His Lys 205 210 215 ctt tct aca gat cac att ccg att ctg tac
aga aca ggc aaa gac aag 845 Leu Ser Thr Asp His Ile Pro Ile Leu Tyr
Arg Thr Gly Lys Asp Lys 220 225 230 aaa gga agc tgc ggt ctt tcc cgt
gtg gac agc acg aca tgc ctg ttc 893 Lys Gly Ser Cys Gly Leu Ser Arg
Val Asp Ser Thr Thr Cys Leu Phe 235 240 245 cct gtt gag gaa aag gca
gtt gaa tat tac ttt gct tct gat gca agt 941 Pro Val Glu Glu Lys Ala
Val Glu Tyr Tyr Phe Ala Ser Asp Ala Ser 250 255 260 gcc gtg ata gag
cac acc aat cgt gtc atc ttt ctg gaa gat gat gat 989 Ala Val Ile Glu
His Thr Asn Arg Val Ile Phe Leu Glu Asp Asp Asp 265 270 275 280 gtt
gca gca gtg gtg gat ggc cgt ctc tct atc cac cga att aaa cga 1037
Val Ala Ala Val Val Asp Gly Arg Leu Ser Ile His Arg Ile Lys Arg 285
290 295 act gca gga gac cat cct ggc cga gct gtg caa act ctc cag atg
gag 1085 Thr Ala Gly Asp His Pro Gly Arg Ala Val Gln Thr Leu Gln
Met Glu 300 305 310 ctc cag cag atc atg aag ggc aac ttt agt tca ttt
atg cag aag gaa 1133 Leu Gln Gln Ile Met Lys Gly Asn Phe Ser Ser
Phe Met Gln Lys Glu 315 320 325 att ttt gag cag cca gaa tct gtt gtg
aac aca atg aga gga aga gtc 1181 Ile Phe Glu Gln Pro Glu Ser Val
Val Asn Thr Met Arg Gly Arg Val 330 335 340 aat ttt gat gac tac act
gtg aat ttg gga ggt ttg aaa gat cac att 1229 Asn Phe Asp Asp Tyr
Thr Val Asn Leu Gly Gly Leu Lys Asp His Ile 345 350 355 360 aag gag
atc cag cgg tgt cgg cgg ttg att ctt att gct tgt ggc aca 1277 Lys
Glu Ile Gln Arg Cys Arg Arg Leu Ile Leu Ile Ala Cys Gly Thr 365 370
375 agt tac cac gct ggt gtg gca acc cgt cag gtc ctg gag gag ctg acc
1325 Ser Tyr His Ala Gly Val Ala Thr Arg Gln Val Leu Glu Glu Leu
Thr 380 385 390 gag ctg ccc gtg atg gtg gag ctt gcc agt gac ttc ttg
gat aga aac 1373 Glu Leu Pro Val Met Val Glu Leu Ala Ser Asp Phe
Leu Asp Arg Asn 395 400 405 act cca gtc ttt cga gat gat gtt tgc ttt
ttc att agt caa tca ggc 1421 Thr Pro Val Phe Arg Asp Asp Val Cys
Phe Phe Ile Ser Gln Ser Gly 410 415 420 gag aca gct gac acc ctg atg
gga ctt cgt tac tgt aag gag aga gga 1469 Glu Thr Ala Asp Thr Leu
Met Gly Leu Arg Tyr Cys Lys Glu Arg Gly 425 430 435 440 gcc tta act
gtg ggg atc aca aat aca gtc ggc agt tct ata tca agg 1517 Ala Leu
Thr Val Gly Ile Thr Asn Thr Val Gly Ser Ser Ile Ser Arg 445 450 455
gag aca gat tgc ggg gtt cat att aat gct ggt cct gag att ggc gtg
1565 Glu Thr Asp Cys Gly Val His Ile Asn Ala Gly Pro Glu Ile Gly
Val 460 465 470 gcc agt aca aag gca tac acc agc cag ttt gtg tcc ctc
gtg atg ttt 1613 Ala Ser Thr Lys Ala Tyr Thr Ser Gln Phe Val Ser
Leu Val Met Phe 475 480 485 gct ctc atg atg tgt gat gac agg atc tcc
atg caa gag aga cgc aaa 1661 Ala Leu Met Met Cys Asp Asp Arg Ile
Ser Met Gln Glu Arg Arg Lys 490 495 500 gag atc atg ctc gga ctg aag
cga ctg ccg gac ttg att aag gaa gtg 1709 Glu Ile Met Leu Gly Leu
Lys Arg Leu Pro Asp Leu Ile Lys Glu Val 505 510 515 520 ctg agc atg
gat gat gaa atc cag aag ctg gcg acg gag ctt tac cac 1757 Leu Ser
Met Asp Asp Glu Ile Gln Lys Leu Ala Thr Glu Leu Tyr His 525 530 535
cag aag tcg gtc ctg ata atg ggg cgg ggc tac cat tat gct aca tgc
1805 Gln Lys Ser Val Leu Ile Met Gly Arg Gly Tyr His Tyr Ala Thr
Cys 540 545 550 ctt gaa ggg gct ctg aaa atc aag gag att act tat atg
cat tcg gaa 1853 Leu Glu Gly Ala Leu Lys Ile Lys Glu Ile Thr Tyr
Met His Ser Glu 555 560 565 ggc atc ctt gct ggt gag ctc aag cac ggc
cct ctg gcc ttg gtg gac 1901 Gly Ile Leu Ala Gly Glu Leu Lys His
Gly Pro Leu Ala Leu Val Asp 570 575 580 aag ttg atg cct gtc atc atg
atc atc atg cga gac cac act tat gcc 1949 Lys Leu Met Pro Val Ile
Met Ile Ile Met Arg Asp His Thr Tyr Ala 585 590 595 600 aag tgc cag
aac gct ctt cag cag gtg gtt gca cgg cag ggg cgt cca 1997 Lys Cys
Gln Asn Ala Leu Gln Gln Val Val Ala Arg Gln Gly Arg Pro 605 610 615
gtc gtg atc tgt gat aag gag gat act gag acc att aag aat aca aaa
2045 Val Val Ile Cys Asp Lys Glu Asp Thr Glu Thr Ile Lys Asn Thr
Lys 620 625 630 agg aca atc aag gtg ccc cac tca gtg gac tgc ttg cag
ggc att ctc 2093 Arg Thr Ile Lys Val Pro His Ser Val Asp Cys Leu
Gln Gly Ile Leu 635 640 645 agt gtg att ccc ctg cag ctg ctg gct ttc
cac ctg gct gtg ctg aga 2141 Ser Val Ile Pro Leu Gln Leu Leu Ala
Phe His Leu Ala Val Leu Arg 650 655 660 ggc tac gat gtt gat ttt cca
cgg aat ctt gcc aaa tct gta aca gta 2189 Gly Tyr Asp Val Asp Phe
Pro Arg Asn Leu Ala Lys Ser Val Thr Val 665 670 675 680 gag
taacagacac ctgaaactta agacagttaa gcaacacgag ataccttttg 2242 Glu
tatttaaatt tttgatttaa actatcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2298
<210> SEQ ID NO 5 <211> LENGTH: 681 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 5 Met
Cys Gly Ile Phe Ala Tyr Leu Asn Tyr His Val Pro Arg Thr Arg 1 5 10
15 Arg Glu Ile Leu Glu Thr Leu Ile Lys Gly Leu Gln Arg Leu Glu Tyr
20 25 30 Arg Gly Tyr Asp Ser Ala Gly Val Gly Leu Asp Gly Gly Asn
Asp Lys 35 40 45 Asp Trp Glu Ala Asn Ala Cys Lys Ile Gln Leu Ile
Lys Lys Lys Gly 50 55 60 Lys Val Lys Ala Leu Asp Glu Glu Val His
Lys Gln Gln Asp Met Asp 65 70 75 80 Leu Asp Ile Glu Phe Asp Val His
Leu Gly Ile Ala His Thr Arg Trp 85 90 95 Ala Thr His Gly Glu Pro
Asn Pro Val Asn Ser His Pro Gln Arg Ser 100 105 110 Asp Lys Asn Asn
Glu Phe Ile Val Ile His Asn Gly Ile Ile Thr Asn 115 120 125 Tyr Lys
Asp Leu Lys Lys Phe Leu Glu Ser Lys Gly Tyr Asp Phe Glu 130 135 140
Ser Glu Thr Asp Thr Glu Thr Ile Ala Lys Leu Val Lys Tyr Met Tyr 145
150 155 160 Asp Asn Trp Glu Ser Gln Asp Val Ser Phe Thr Thr Leu Val
Glu Arg 165 170 175 Val Ile Gln Gln Leu Glu Gly Ala Phe Ala Leu Val
Phe Lys Ser Val 180 185 190 His Phe Pro Gly Gln Ala Val Gly Thr Arg
Arg Gly Ser Pro Leu Leu 195 200 205 Ile Gly Val Arg Ser Glu His Lys
Leu Ser Thr Asp His Ile Pro Ile 210 215 220 Leu Tyr Arg Thr Gly Lys
Asp Lys Lys Gly Ser Cys Gly Leu Ser Arg 225 230 235 240 Val Asp Ser
Thr Thr Cys Leu Phe Pro Val Glu Glu Lys Ala Val Glu 245 250 255 Tyr
Tyr Phe Ala Ser Asp Ala Ser Ala Val Ile Glu His Thr Asn Arg 260 265
270 Val Ile Phe Leu Glu Asp Asp Asp Val Ala Ala Val Val Asp Gly Arg
275 280 285 Leu Ser Ile His Arg Ile Lys Arg Thr Ala Gly Asp His Pro
Gly Arg 290 295 300 Ala Val Gln Thr Leu Gln Met Glu Leu Gln Gln Ile
Met Lys Gly Asn 305 310 315 320 Phe Ser Ser Phe Met Gln Lys Glu Ile
Phe Glu Gln Pro Glu Ser Val 325 330 335 Val Asn Thr Met Arg Gly Arg
Val Asn Phe Asp Asp Tyr Thr Val Asn 340 345 350 Leu Gly Gly Leu Lys
Asp His Ile Lys Glu Ile Gln Arg Cys Arg Arg 355 360 365 Leu Ile Leu
Ile Ala Cys Gly Thr Ser Tyr His Ala Gly Val Ala Thr 370 375 380 Arg
Gln Val Leu Glu Glu Leu Thr Glu Leu Pro Val Met Val Glu Leu 385 390
395 400 Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp Asp
Val 405 410 415 Cys Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr
Leu Met Gly 420 425 430 Leu Arg Tyr Cys Lys Glu Arg Gly Ala Leu Thr
Val Gly Ile Thr Asn 435 440 445 Thr Val Gly Ser Ser Ile Ser Arg Glu
Thr Asp Cys Gly Val His Ile 450 455 460 Asn Ala Gly Pro Glu Ile Gly
Val Ala Ser Thr Lys Ala Tyr Thr Ser 465 470 475 480 Gln Phe Val Ser
Leu Val Met Phe Ala Leu Met Met Cys Asp Asp Arg 485 490 495 Ile Ser
Met Gln Glu Arg Arg Lys Glu Ile Met Leu Gly Leu Lys Arg 500 505 510
Leu Pro Asp Leu Ile Lys Glu Val Leu Ser Met Asp Asp Glu Ile Gln 515
520 525 Lys Leu Ala Thr Glu Leu Tyr His Gln Lys Ser Val Leu Ile Met
Gly 530 535 540 Arg Gly Tyr His Tyr Ala Thr Cys Leu Glu Gly Ala Leu
Lys Ile Lys 545 550 555 560 Glu Ile Thr Tyr Met His Ser Glu Gly Ile
Leu Ala Gly Glu Leu Lys 565 570 575 His Gly Pro Leu Ala Leu Val Asp
Lys Leu Met Pro Val Ile Met Ile 580 585 590 Ile Met Arg Asp His Thr
Tyr Ala Lys Cys Gln Asn Ala Leu Gln Gln 595 600 605 Val Val Ala Arg
Gln Gly Arg Pro Val Val Ile Cys Asp Lys Glu Asp 610 615 620 Thr Glu
Thr Ile Lys Asn Thr Lys Arg Thr Ile Lys Val Pro His Ser 625 630 635
640 Val Asp Cys Leu Gln Gly Ile Leu Ser Val Ile Pro Leu Gln Leu Leu
645 650 655 Ala Phe His Leu Ala Val Leu Arg Gly Tyr Asp Val Asp Phe
Pro Arg 660 665 670 Asn Leu Ala Lys Ser Val Thr Val Glu 675 680
<210> SEQ ID NO 6 <211> LENGTH: 2049 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(2046)
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: BC031928.1 <309> DATABASE ENTRY DATE: 2003-10-07
<313> RELEVANT RESIDUES IN SEQ ID NO: (51)..(299) <400>
SEQUENCE: 6 atg tgc gga atc ttt gcc tac atg aat tac aga gtt ccc aag
aca agg 48 Met Cys Gly Ile Phe Ala Tyr Met Asn Tyr Arg Val Pro Lys
Thr Arg 1 5 10 15 aaa gag att ttc gaa acc ctt atc agg ggt ctg cag
cgg ctg gag tac 96 Lys Glu Ile Phe Glu Thr Leu Ile Arg Gly Leu Gln
Arg Leu Glu Tyr 20 25 30 cgg ggc tat gac tct gcg ggg gtt gcc att
gat ggg aat aac cac gaa 144 Arg Gly Tyr Asp Ser Ala Gly Val Ala Ile
Asp Gly Asn Asn His Glu 35 40 45 gtc aaa gaa aga cac atc cat ctt
gtg aag aaa agg ggg aaa gta aag 192 Val Lys Glu Arg His Ile His Leu
Val Lys Lys Arg Gly Lys Val Lys 50 55 60 gct ctg gat gaa gaa ctt
tac aag caa gat agc atg gac ttg aag gtg 240 Ala Leu Asp Glu Glu Leu
Tyr Lys Gln Asp Ser Met Asp Leu Lys Val 65 70 75 80 gag ttt gag aca
cac ttc ggc att gcc cac aca cgt tgg gcc acc cac 288 Glu Phe Glu Thr
His Phe Gly Ile Ala His Thr Arg Trp Ala Thr His 85 90 95 ggg gtt
ccc aat gct gtc aac agt cac ccg cag cgt tcg gac aaa gac 336 Gly Val
Pro Asn Ala Val Asn Ser His Pro Gln Arg Ser Asp Lys Asp 100 105 110
aat gaa ttt gtt gtc atc cac aac ggg atc atc act aat tac aag gat 384
Asn Glu Phe Val Val Ile His Asn Gly Ile Ile Thr Asn Tyr Lys Asp 115
120 125 cta agg aag ttt ctg gaa agc aaa ggc tac gag ttt gag tca gaa
aca 432 Leu Arg Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe Glu Ser Glu
Thr 130 135 140 gac acg gag acc atc gcc aag ctg att aaa tat gta ttt
gac aac aga 480 Asp Thr Glu Thr Ile Ala Lys Leu Ile Lys Tyr Val Phe
Asp Asn Arg 145 150 155 160 gag act gag gac ata acg ttt tcc aca ttg
gtc gaa aga gtc att cag 528 Glu Thr Glu Asp Ile Thr Phe Ser Thr Leu
Val Glu Arg Val Ile Gln 165 170 175 cag ttg gaa ggc gcc ttt gca ctg
gtt ttc aag agt att cac tac ccg 576 Gln Leu Glu Gly Ala Phe Ala Leu
Val Phe Lys Ser Ile His Tyr Pro 180 185 190 gga gaa gct gtc gcc acg
agg aga ggc agc ccc ttg ctc atc ggg gta 624 Gly Glu Ala Val Ala Thr
Arg Arg Gly Ser Pro Leu Leu Ile Gly Val 195 200 205 cga agc aaa tac
aaa ctc tcc aca gag cag atc ccc gtc tta tat ccg 672 Arg Ser Lys Tyr
Lys Leu Ser Thr Glu Gln Ile Pro Val Leu Tyr Pro 210 215 220 aca tgc
aat atc gag aat gtg aag aat atc tgc aag act agg atg aag 720 Thr Cys
Asn Ile Glu Asn Val Lys Asn Ile Cys Lys Thr Arg Met Lys 225 230 235
240 aga ctg gac agc tcc acc tgc ctg cac gct gtg ggc gat aaa gct gtg
768 Arg Leu Asp Ser Ser Thr Cys Leu His Ala Val Gly Asp Lys Ala Val
245 250 255 gaa ttc ttc ttt gct tct gat gca agt gcc atc ata gaa cac
acc aac 816 Glu Phe Phe Phe Ala Ser Asp Ala Ser Ala Ile Ile Glu His
Thr Asn 260 265 270 cgg gtc atc ttc tta gaa gat gat gat atc gct gca
gtg gct gat ggg 864 Arg Val Ile Phe Leu Glu Asp Asp Asp Ile Ala Ala
Val Ala Asp Gly 275 280 285 aaa ctc tcc att cac cga gtc aag cgc tca
gct act gat gac ccc tcc 912 Lys Leu Ser Ile His Arg Val Lys Arg Ser
Ala Thr Asp Asp Pro Ser 290 295 300 cga gcc atc cag acc ttg cag atg
gaa ctg cag caa ata atg aaa ggt 960 Arg Ala Ile Gln Thr Leu Gln Met
Glu Leu Gln Gln Ile Met Lys Gly 305 310 315 320 aac ttc agc gca ttt
atg cag aag gag atc ttc gag cag cca gaa tca 1008 Asn Phe Ser Ala
Phe Met Gln Lys Glu Ile Phe Glu Gln Pro Glu Ser 325 330 335 gtt ttt
aat acc atg aga ggt cgg gtg aat ttt gag acc aac aca gtg 1056 Val
Phe Asn Thr Met Arg Gly Arg Val Asn Phe Glu Thr Asn Thr Val 340 345
350 ctc ctg ggt ggc ttg aag gac cat ttg aaa gag atc cga cga tgc cga
1104 Leu Leu Gly Gly Leu Lys Asp His Leu Lys Glu Ile Arg Arg Cys
Arg 355 360 365 agg ctc att gtg att ggc tgt gga acc agc tac cat gcc
gct gtg gct 1152 Arg Leu Ile Val Ile Gly Cys Gly Thr Ser Tyr His
Ala Ala Val Ala 370 375 380 aca cgg caa gtc tta gag gaa ctg acc gag
ctg cct gtg atg gtt gaa 1200 Thr Arg Gln Val Leu Glu Glu Leu Thr
Glu Leu Pro Val Met Val Glu 385 390 395 400 ctt gcc agt gac ttt ctg
gac agg aac aca cct gtg ttc agg gat gac 1248 Leu Ala Ser Asp Phe
Leu Asp Arg Asn Thr Pro Val Phe Arg Asp Asp 405 410 415 gtt tgc ttt
ttc ata agc caa tca ggt gag act gca gac acg ctc ctg 1296 Val Cys
Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr Leu Leu 420 425 430
gcg ctg cga tac tgt aag gat cga ggt gcg ctg acc gtg ggc atc acc
1344 Ala Leu Arg Tyr Cys Lys Asp Arg Gly Ala Leu Thr Val Gly Ile
Thr 435 440 445 aac acc gtg ggt agc tcc atc tcc cgg gag act gac tgt
ggc gtc cac 1392 Asn Thr Val Gly Ser Ser Ile Ser Arg Glu Thr Asp
Cys Gly Val His 450 455 460 atc aac gca ggg ccc gag att ggg gtg gcc
agc acc aag gcg tac acc 1440 Ile Asn Ala Gly Pro Glu Ile Gly Val
Ala Ser Thr Lys Ala Tyr Thr 465 470 475 480 agc cag ttc atc tct ctg
gtg atg ttt ggt ttg atg atg tct gaa gat 1488 Ser Gln Phe Ile Ser
Leu Val Met Phe Gly Leu Met Met Ser Glu Asp 485 490 495 cga att tct
cta cag aac agg aga caa gag atc atc cgt ggc ctc aga 1536 Arg Ile
Ser Leu Gln Asn Arg Arg Gln Glu Ile Ile Arg Gly Leu Arg 500 505 510
tct tta ccg gag ctg atc aaa gaa gtg ctg tcc ctg gat gag aag atc
1584 Ser Leu Pro Glu Leu Ile Lys Glu Val Leu Ser Leu Asp Glu Lys
Ile 515 520 525 cat gac ttg gcc ctg gag ctc tac aca caa agg tct ctc
ctc gtg atg 1632 His Asp Leu Ala Leu Glu Leu Tyr Thr Gln Arg Ser
Leu Leu Val Met 530 535 540 gga cgg gga tat aac tat gcc aca tgt ctg
gaa ggt gcc ttg aaa att 1680 Gly Arg Gly Tyr Asn Tyr Ala Thr Cys
Leu Glu Gly Ala Leu Lys Ile 545 550 555 560 aag gag ata acc tac atg
cat tca gaa ggt atc cta gcc gga gag ctg 1728 Lys Glu Ile Thr Tyr
Met His Ser Glu Gly Ile Leu Ala Gly Glu Leu 565 570 575 aag cac ggg
ccc ctt gct ctc gtc gac aag cag atg cca gtc atc atg 1776 Lys His
Gly Pro Leu Ala Leu Val Asp Lys Gln Met Pro Val Ile Met 580 585 590
gtc atc atg aag gat cct tgc ttt gcc aag tgc cag aat gcc ctg cag
1824 Val Ile Met Lys Asp Pro Cys Phe Ala Lys Cys Gln Asn Ala Leu
Gln 595 600 605 cag gtc act gcc cgc cag ggt cgc cca atc ata ctg tgt
tcc aag gat 1872 Gln Val Thr Ala Arg Gln Gly Arg Pro Ile Ile Leu
Cys Ser Lys Asp 610 615 620 gac acc gag agc tcc aag ttt gca tat aaa
acc att gaa ctt ccc cac 1920 Asp Thr Glu Ser Ser Lys Phe Ala Tyr
Lys Thr Ile Glu Leu Pro His 625 630 635 640 aca gtg gac tgt ctc cag
ggt atc ctg agc gtg att cca ctc cag ctt 1968 Thr Val Asp Cys Leu
Gln Gly Ile Leu Ser Val Ile Pro Leu Gln Leu 645 650 655 ctg tcc ttc
cac ctg gct gtc ctc cga ggt tat gat gtt gac ttc ccc 2016 Leu Ser
Phe His Leu Ala Val Leu Arg Gly Tyr Asp Val Asp Phe Pro 660 665 670
aga aac cta gcc aag tct gtc act gtg gaa tga 2049 Arg Asn Leu Ala
Lys Ser Val Thr Val Glu 675 680 <210> SEQ ID NO 7 <211>
LENGTH: 682 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 7 Met Cys Gly Ile Phe Ala Tyr Met
Asn Tyr Arg Val Pro Lys Thr Arg 1 5 10 15 Lys Glu Ile Phe Glu Thr
Leu Ile Arg Gly Leu Gln Arg Leu Glu Tyr 20 25 30 Arg Gly Tyr Asp
Ser Ala Gly Val Ala Ile Asp Gly Asn Asn His Glu 35 40 45 Val Lys
Glu Arg His Ile His Leu Val Lys Lys Arg Gly Lys Val Lys 50 55 60
Ala Leu Asp Glu Glu Leu Tyr Lys Gln Asp Ser Met Asp Leu Lys Val 65
70 75 80 Glu Phe Glu Thr His Phe Gly Ile Ala His Thr Arg Trp Ala
Thr His 85 90 95 Gly Val Pro Asn Ala Val Asn Ser His Pro Gln Arg
Ser Asp Lys Asp 100 105 110 Asn Glu Phe Val Val Ile His Asn Gly Ile
Ile Thr Asn Tyr Lys Asp 115 120 125 Leu Arg Lys Phe Leu Glu Ser Lys
Gly Tyr Glu Phe Glu Ser Glu Thr 130 135 140 Asp Thr Glu Thr Ile Ala
Lys Leu Ile Lys Tyr Val Phe Asp Asn Arg 145 150 155 160 Glu Thr Glu
Asp Ile Thr Phe Ser Thr Leu Val Glu Arg Val Ile Gln 165 170 175 Gln
Leu Glu Gly Ala Phe Ala Leu Val Phe Lys Ser Ile His Tyr Pro 180 185
190 Gly Glu Ala Val Ala Thr Arg Arg Gly Ser Pro Leu Leu Ile Gly Val
195 200 205 Arg Ser Lys Tyr Lys Leu Ser Thr Glu Gln Ile Pro Val Leu
Tyr Pro 210 215 220 Thr Cys Asn Ile Glu Asn Val Lys Asn Ile Cys Lys
Thr Arg Met Lys 225 230 235 240 Arg Leu Asp Ser Ser Thr Cys Leu His
Ala Val Gly Asp Lys Ala Val 245 250 255 Glu Phe Phe Phe Ala Ser Asp
Ala Ser Ala Ile Ile Glu His Thr Asn 260 265 270 Arg Val Ile Phe Leu
Glu Asp Asp Asp Ile Ala Ala Val Ala Asp Gly 275 280 285 Lys Leu Ser
Ile His Arg Val Lys Arg Ser Ala Thr Asp Asp Pro Ser 290 295 300 Arg
Ala Ile Gln Thr Leu Gln Met Glu Leu Gln Gln Ile Met Lys Gly 305 310
315 320 Asn Phe Ser Ala Phe Met Gln Lys Glu Ile Phe Glu Gln Pro Glu
Ser 325 330 335 Val Phe Asn Thr Met Arg Gly Arg Val Asn Phe Glu Thr
Asn Thr Val 340 345 350 Leu Leu Gly Gly Leu Lys Asp His Leu Lys Glu
Ile Arg Arg Cys Arg 355 360 365 Arg Leu Ile Val Ile Gly Cys Gly Thr
Ser Tyr His Ala Ala Val Ala 370 375 380 Thr Arg Gln Val Leu Glu Glu
Leu Thr Glu Leu Pro Val Met Val Glu 385 390 395 400 Leu Ala Ser Asp
Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp Asp 405 410 415 Val Cys
Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr Leu Leu 420 425 430
Ala Leu Arg Tyr Cys Lys Asp Arg Gly Ala Leu Thr Val Gly Ile Thr 435
440 445 Asn Thr Val Gly Ser Ser Ile Ser Arg Glu Thr Asp Cys Gly Val
His 450 455 460 Ile Asn Ala Gly Pro Glu Ile Gly Val Ala Ser Thr Lys
Ala Tyr Thr 465 470 475 480 Ser Gln Phe Ile Ser Leu Val Met Phe Gly
Leu Met Met Ser Glu Asp 485 490 495 Arg Ile Ser Leu Gln Asn Arg Arg
Gln Glu Ile Ile Arg Gly Leu Arg 500 505 510 Ser Leu Pro Glu Leu Ile
Lys Glu Val Leu Ser Leu Asp Glu Lys Ile 515 520 525 His Asp Leu Ala
Leu Glu Leu Tyr Thr Gln Arg Ser Leu Leu Val Met 530 535 540 Gly Arg
Gly Tyr Asn Tyr Ala Thr Cys Leu Glu Gly Ala Leu Lys Ile 545 550 555
560 Lys Glu Ile Thr Tyr Met His Ser Glu Gly Ile Leu Ala Gly Glu Leu
565 570 575 Lys His Gly Pro Leu Ala Leu Val Asp Lys Gln Met Pro Val
Ile Met 580 585 590 Val Ile Met Lys Asp Pro Cys Phe Ala Lys Cys Gln
Asn Ala Leu Gln 595 600 605 Gln Val Thr Ala Arg Gln Gly Arg Pro Ile
Ile Leu Cys Ser Lys Asp 610 615 620 Asp Thr Glu Ser Ser Lys Phe Ala
Tyr Lys Thr Ile Glu Leu Pro His 625 630 635 640 Thr Val Asp Cys Leu
Gln Gly Ile Leu Ser Val Ile Pro Leu Gln Leu 645 650 655 Leu Ser Phe
His Leu Ala Val Leu Arg Gly Tyr Asp Val Asp Phe Pro 660 665 670 Arg
Asn Leu Ala Lys Ser Val Thr Val Glu 675 680 <210> SEQ ID NO 8
<211> LENGTH: 1830 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(1827) <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
U00096.2 <309> DATABASE ENTRY DATE: 2005-09-08 <313>
RELEVANT RESIDUES IN SEQ ID NO: (3909862)..(3911691) <400>
SEQUENCE: 8 atg tgt gga att gtt ggc gcg atc gcg caa cgt gat gta gca
gaa atc 48 Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala
Glu Ile 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga
tat gac tct gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly
Tyr Asp Ser Ala 20 25 30 ggt ctg gcc gtt gtt gat gca gaa ggt cat
atg acc cgc ctg cgt cgc 144 Gly Leu Ala Val Val Asp Ala Glu Gly His
Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gca
cag gca gcg gaa gaa cat cct ctg 192 Leu Gly Lys Val Gln Met Leu Ala
Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att
gct cac act cgc tgg gcg acc cac ggt gaa 240 His Gly Gly Thr Gly Ile
Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 cct tca gaa gtg
aat gcg cat ccg cat gtt tct gaa cac att gtg gtg 288 Pro Ser Glu Val
Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95 gtg cat
aac ggc atc atc gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His
Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110
aaa gcg cgt ggc tat acc ttc gtt tct gaa acc gac acc gaa gtg att 384
Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115
120 125 gcc cat ctg gtg aac tgg gag ctg aaa caa ggc ggg act ctg cgt
gag 432 Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg
Glu 130 135 140 gcc gtt ctg cgt gct atc ccg cag ctg cgt ggt gcg tac
ggt aca gtg 480 Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr
Gly Thr Val 145 150 155 160 atc atg gac tcc cgt cac ccg gat acc ctg
ctg gcg gca cgt tct ggt 528 Ile Met Asp Ser Arg His Pro Asp Thr Leu
Leu Ala Ala Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg
atg ggc gaa aac ttt atc gct tct 576 Ser Pro Leu Val Ile Gly Leu Gly
Met Gly Glu Asn Phe Ile Ala Ser 180 185 190 gac cag ctg gcg ctg ttg
ccg gtg acc cgt cgc ttt atc ttc ctt gaa 624 Asp Gln Leu Ala Leu Leu
Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205 gag ggc gat att
gcg gaa atc act cgc cgt tcg gta aac atc ttc gat 672 Glu Gly Asp Ile
Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220 aaa act
ggc gcg gaa gta aaa cgt cag gat atc gaa tcc aat ctg caa 720 Lys Thr
Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln 225 230 235
240 tat gac gcg ggc gat aaa ggc att tac cgt cac tac atg cag aaa gag
768 Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu
245 250 255 atc tac gaa cag ccg aac gcg atc aaa aac acc ctt acc gga
cgc atc 816 Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly
Arg Ile 260 265 270 agc cac ggt cag gtt gat tta agc gag ctg gga ccg
aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro
Asn Ala Asp Glu 275 280 285 ctg ctg tcg aag gtt gag cat att cag atc
ctc gcc tgt ggt act tct 912 Leu Leu Ser Lys Val Glu His Ile Gln Ile
Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tcc ggt atg gtt tcc cgc
tac tgg ttt gaa tcg cta gca ggt 960 Tyr Asn Ser Gly Met Val Ser Arg
Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 att ccg tgc gac gtc
gaa atc gcc tct gaa ttc cgc tat cgc aaa tct 1008 Ile Pro Cys Asp
Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg
cgt cgt aac agc ctg atg atc acc ttg tca cag tct ggc gaa 1056 Ala
Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345
350 acc gcg gat acc ctg gct ggc ctg cgt ctg tcg aaa gag ctg ggt tac
1104 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly
Tyr 355 360 365 ctt ggt tca ctg gca atc tgt aac gtt ccg ggt tct tct
ctg gtg cgc 1152 Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser
Ser Leu Val Arg 370 375 380 gaa tcc gat ctg gcg cta atg acc aac gcg
ggt aca gaa atc ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn
Ala Gly Thr Glu Ile Gly Val 385 390 395 400 gca tcc act aaa gca ttc
acc act cag tta act gtg ctg ttg atg ctg 1248 Ala Ser Thr Lys Ala
Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag
ctg tct cgc ctg aaa ggt ctg gat gcc tcc att gaa cat 1296 Val Ala
Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430
gac atc gtg cat ggt ctg cag gcg ctg ccg agc cgt att gag cag atg
1344 Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln
Met 435 440 445 ctg tct cag gac aaa cgc att gaa gcg ctg gca gaa gat
ttc tct gac 1392 Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu
Asp Phe Ser Asp 450 455 460 aaa cat cac gcg ctg ttc ctg ggc cgt ggc
gat cag tac cca atc gcg 1440 Lys His His Ala Leu Phe Leu Gly Arg
Gly Asp Gln Tyr Pro Ile Ala 465 470 475 480 ctg gaa ggc gca ttg aag
ttg aaa gag atc tct tac att cac gct gaa 1488 Leu Glu Gly Ala Leu
Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495 gcc tac gct
gct ggc gaa ctg aaa cac ggt ccg ctg gcg cta att gat 1536 Ala Tyr
Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510
gcc gat atg ccg gtt att gtt gtt gca ccg aac aac gaa ttg ctg gaa
1584 Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu
Glu 515 520 525 aaa ctg aaa tcc aac att gaa gaa gtt cgc gcg cgt ggc
ggt cag ttg 1632 Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg
Gly Gly Gln Leu 530 535 540 tat gtc ttc gcc gat cag gat gcg ggt ttt
gta agt agc gat aac atg 1680 Tyr Val Phe Ala Asp Gln Asp Ala Gly
Phe Val Ser Ser Asp Asn Met 545 550 555 560 cac atc atc gag atg ccg
cat gtg gaa gag gtg att gca ccg atc ttc 1728 His Ile Ile Glu Met
Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575 tac acc gtt
ccg ctg cag ctg ctg gct tac cat gtc gcg ctg atc aaa 1776 Tyr Thr
Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590
ggc acc gac gtt gac cag ccg cgt aac ctg gca aaa tcg gtt acg gtt
1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr
Val 595 600 605 gag taa 1830 Glu <210> SEQ ID NO 9
<211> LENGTH: 609 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 9 Met Cys Gly Ile Val Gly
Ala Ile Ala Gln Arg Asp Val Ala Glu Ile 1 5 10 15 Leu Leu Glu Gly
Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Leu
Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45
Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50
55 60 His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly
Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His
Ile Val Val 85 90 95 Val His Asn Gly Ile Ile Glu Asn His Glu Pro
Leu Arg Glu Glu Leu 100 105 110 Lys Ala Arg Gly Tyr Thr Phe Val Ser
Glu Thr Asp Thr Glu Val Ile 115 120 125 Ala His Leu Val Asn Trp Glu
Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Ala Val Leu Arg Ala
Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 Ile Met
Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175
Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180
185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu
Glu 195 200 205 Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn
Ile Phe Asp 210 215 220 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile
Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Ala Gly Asp Lys Gly Ile
Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 Ile Tyr Glu Gln Pro Asn
Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270 Ser His Gly Gln
Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 Leu Leu
Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300
Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305
310 315 320 Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg
Lys Ser 325 330 335 Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser
Gln Ser Gly Glu 340 345 350 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu
Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala Ile Cys Asn
Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Ser Asp Leu Ala Leu
Met Thr Asn Ala Gly Thr Glu Ile Gly Val 385 390 395 400 Ala Ser Thr
Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val
Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425
430 Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met
435 440 445 Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe
Ser Asp 450 455 460 Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln
Tyr Pro Ile Ala 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu
Ile Ser Tyr Ile His Ala Glu 485 490 495 Ala Tyr Ala Ala Gly Glu Leu
Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510 Ala Asp Met Pro Val
Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys
Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu 530 535 540 Tyr
Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met 545 550
555 560 His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile
Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala
Leu Ile Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala
Lys Ser Val Thr Val 595 600 605 Glu <210> SEQ ID NO 10
<211> LENGTH: 1830 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic sequence encoding an Escherichia coli
protein having the activity of a GFAT <400> SEQUENCE: 10
atgtgcggaa ttgttggtgc tatcgcccaa agagacgttg ctgagatttt gttagagggt
60 ctgcgaaggc tagagtatag aggatatgac tccgctggtc tggctgtcgt
tgatgctgag 120 ggtcatatga caaggctaag aaggttagga aaggttcaga
tgcttgctca ggcagctgag 180 gaacatccat tgcatggagg tactggtatt
gcacatacca ggtgggctac tcatggggag 240 ccatcagaag ttaatgctca
tccacatgtg agtgagcata tcgttgtagt tcacaatggg 300 ataattgaaa
accacgaacc attgagggaa gagttaaagg caagaggata tacttttgtg 360
agtgagactg acactgaggt tattgcacat ttagtgaact gggaactcaa acaggggggc
420 acattgcgtg aggctgtgtt aagagctatt cctcaactta gaggtgcata
cggtactgtt 480 attatggatt caagacaccc agatactctc cttgcagcta
gatcaggtag tcccttggtc 540 ataggacttg gaatgggtga aaattttatc
gctagcgacc aattggcctt attgccagtt 600 acaagacgat ttattttcct
tgaagagggc gatattgctg agattactag aaggtctgtg 660 aacatctttg
ataagactgg cgctgaggtt aaacgtcagg atatcgagtc taaccttcaa 720
tacgatgctg gtgataaagg aatttacagg cattatatgc aaaaggaaat ttatgaacaa
780 ccaaatgcta tcaaaaacac acttactggc cgtatttctc atggacaggt
cgatttaagc 840 gagcttggtc ctaatgcaga cgaactgcta tcaaaagttg
agcacataca gatactggca 900 tgcggaacta gttataattc aggaatggtc
tctagatact ggttcgaaag cttggcaggt 960 ataccttgtg atgtagagat
cgcttctgag tttaggtata gaaagtctgc tgtgcgtaga 1020 aattcattaa
tgattacatt atctcaatcc ggagaaacag cagatacact ggctggattg 1080
aggctttcta aggaactcgg atatctgggt tcacttgcta tttgtaatgt accaggttcc
1140 tcattggttc gtgaatcaga tctagcactt atgacaaatg caggaactga
aataggtgtg 1200 gcaagtacca aggctttcac aacccaactg accgtacttt
taatgttggt agcaaaactc 1260 agtcgattaa aggggctaga tgcatctatc
gaacatgata ttgttcacgg gcttcaagct 1320 ctcccttcaa gaattgaaca
aatgctttca caagataaga gaatagaggc attggctgaa 1380 gatttttccg
acaaacatca cgcattgttt cttggacgtg gcgatcaata tccaattgca 1440
ttggaaggag ctttgaagtt gaaagaaata agttacattc acgcagaagc atatgcagct
1500 ggagaactca agcatggtcc tttggcactc atcgacgctg acatgcccgt
gatcgtagtg 1560 gctcctaata acgaactgct cgaaaagctt aaatcaaata
tcgaagaggt tcgagctaga 1620 ggaggtcagc tttacgtttt cgctgaacaa
gatgctggat tcgtgtcaag cgataatatg 1680 catataattg aaatgcctca
cgttgaagaa gtgattgcac ctatatttta tacagtccca 1740 ttgcaacttc
tagcttacca tgttgcactt attaaaggaa ctgatgttga tcagcctaga 1800
aacctagcaa aatctgtaac agtcgaataa 1830 <210> SEQ ID NO 11
<211> LENGTH: 1260 <212> TYPE: DNA <213>
ORGANISM: Paramecium bursaria Chlorella Virus 1 <220>
FEATURE: <221> NAME/KEY: CDS <222> LOCATION:
(62)..(1228) <300> PUBLICATION INFORMATION: <308>
DATABASE ACCESSION NUMBER: U42580.4 <309> DATABASE ENTRY
DATE: 2004-09-20 <313> RELEVANT RESIDUES IN SEQ ID NO:
(291749.)..(292918) <400> SEQUENCE: 11 atcaacgtga tttatatttt
aaacaaagac cattcacatc tttagtactt aattaattat 60 a atg tca cga atc
gca gtc gtt ggt tgt ggt tac gtc gga acc gct tgt 109 Met Ser Arg Ile
Ala Val Val Gly Cys Gly Tyr Val Gly Thr Ala Cys 1 5 10 15 gca gta
ctt ctt gct caa aaa aac gaa gtc atc gtg ctt gat att agc 157 Ala Val
Leu Leu Ala Gln Lys Asn Glu Val Ile Val Leu Asp Ile Ser 20 25 30
gaa gac cgt gtt caa cta atc aag aac aag aag agt cca atc gag gac 205
Glu Asp Arg Val Gln Leu Ile Lys Asn Lys Lys Ser Pro Ile Glu Asp 35
40 45 aag gaa atc gaa gag ttt ctc gaa acg aaa gac ctg aac ctg acc
gcg 253 Lys Glu Ile Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn Leu Thr
Ala 50 55 60 acg act gac aag gtt ctt gca tac gaa aac gcc gaa ttt
gtc atc atc 301 Thr Thr Asp Lys Val Leu Ala Tyr Glu Asn Ala Glu Phe
Val Ile Ile 65 70 75 80 gca acc ccg act gac tat gac gtg gtt act agg
tat ttt aac acg aaa 349 Ala Thr Pro Thr Asp Tyr Asp Val Val Thr Arg
Tyr Phe Asn Thr Lys 85 90 95 tct gtg gaa aac gtc att ggg gac gtg
atc aaa aat aca cag acc cat 397 Ser Val Glu Asn Val Ile Gly Asp Val
Ile Lys Asn Thr Gln Thr His 100 105 110 cca act atc gtg att aaa tct
acc atc ccc att gga ttt gtt gat aag 445 Pro Thr Ile Val Ile Lys Ser
Thr Ile Pro Ile Gly Phe Val Asp Lys 115 120 125 gtt cgt gag caa ttc
gac tac caa aat atc att ttc tcc cca gaa ttt 493 Val Arg Glu Gln Phe
Asp Tyr Gln Asn Ile Ile Phe Ser Pro Glu Phe 130 135 140 ctg cgt gaa
ggt aga gcc ttg tat gat aat ctc tac cca tcc cgt atc 541 Leu Arg Glu
Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile 145 150 155 160
atc gta gga gat gat tcc ccc att gcg ctt aag ttc gca aac ctt ctc 589
Ile Val Gly Asp Asp Ser Pro Ile Ala Leu Lys Phe Ala Asn Leu Leu 165
170 175 gtt gaa ggt tct aaa act ccg ctt gcc cct gtc ctg acg atg gga
act 637 Val Glu Gly Ser Lys Thr Pro Leu Ala Pro Val Leu Thr Met Gly
Thr 180 185 190 cgc gaa gcc gag gcc gtc aaa cta ttc tct aac acg tat
ctt gca atg 685 Arg Glu Ala Glu Ala Val Lys Leu Phe Ser Asn Thr Tyr
Leu Ala Met 195 200 205 cga gtt gca tac ttc aac gaa cta gat aca ttc
gca atg tct cac ggt 733 Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr Phe
Ala Met Ser His Gly 210 215 220 atg aat gcg aaa gaa atc att gat ggt
gtg act ctg gag cct cgc att 781 Met Asn Ala Lys Glu Ile Ile Asp Gly
Val Thr Leu Glu Pro Arg Ile 225 230 235 240 ggt cag ggg tac tca aac
cct tcg ttc ggt tat gga gct tat tgc ttt 829 Gly Gln Gly Tyr Ser Asn
Pro Ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255 cca aag gat acg
aag caa ctg ctg gct aat ttc gag gga gtg cct caa 877 Pro Lys Asp Thr
Lys Gln Leu Leu Ala Asn Phe Glu Gly Val Pro Gln 260 265 270 gat atc
atc gga gca att gta gaa tca aat gag act cgc aag gaa gtg 925 Asp Ile
Ile Gly Ala Ile Val Glu Ser Asn Glu Thr Arg Lys Glu Val 275 280 285
att gtg agt gaa gta gaa aat cgt ttc ccc acg act gtt ggt gtg tat 973
Ile Val Ser Glu Val Glu Asn Arg Phe Pro Thr Thr Val Gly Val Tyr 290
295 300 aag ctc gcc gct aaa gcg ggt tct gat aat ttt cgg agt tct gca
att 1021 Lys Leu Ala Ala Lys Ala Gly Ser Asp Asn Phe Arg Ser Ser
Ala Ile 305 310 315 320 gta gac ata atg gag cga ctt gca aac aag ggt
tat cac att aag att 1069 Val Asp Ile Met Glu Arg Leu Ala Asn Lys
Gly Tyr His Ile Lys Ile 325 330 335 ttc gaa cca act gtg gaa caa ttc
gaa aac ttt gaa gtt gat aac aac 1117 Phe Glu Pro Thr Val Glu Gln
Phe Glu Asn Phe Glu Val Asp Asn Asn 340 345 350 ctg aca aca ttt gcg
act gag agc gat gta att atc gca aac aga gtt 1165 Leu Thr Thr Phe
Ala Thr Glu Ser Asp Val Ile Ile Ala Asn Arg Val 355 360 365 ccc gtt
gaa cat cgc att ctc ttt ggt aaa aaa tta atc aca cgt gat 1213 Pro
Val Glu His Arg Ile Leu Phe Gly Lys Lys Leu Ile Thr Arg Asp 370 375
380 gta tat ggc gat aac taaaatgttt tcaatatgat gttgttaatg at 1260
Val Tyr Gly Asp Asn 385 <210> SEQ ID NO 12 <211>
LENGTH: 389 <212> TYPE: PRT <213> ORGANISM: Paramecium
bursaria Chlorella Virus 1 <400> SEQUENCE: 12 Met Ser Arg Ile
Ala Val Val Gly Cys Gly Tyr Val Gly Thr Ala Cys 1 5 10 15 Ala Val
Leu Leu Ala Gln Lys Asn Glu Val Ile Val Leu Asp Ile Ser 20 25 30
Glu Asp Arg Val Gln Leu Ile Lys Asn Lys Lys Ser Pro Ile Glu Asp 35
40 45 Lys Glu Ile Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn Leu Thr
Ala 50 55 60 Thr Thr Asp Lys Val Leu Ala Tyr Glu Asn Ala Glu Phe
Val Ile Ile 65 70 75 80 Ala Thr Pro Thr Asp Tyr Asp Val Val Thr Arg
Tyr Phe Asn Thr Lys 85 90 95 Ser Val Glu Asn Val Ile Gly Asp Val
Ile Lys Asn Thr Gln Thr His 100 105 110 Pro Thr Ile Val Ile Lys Ser
Thr Ile Pro Ile Gly Phe Val Asp Lys 115 120 125 Val Arg Glu Gln Phe
Asp Tyr Gln Asn Ile Ile Phe Ser Pro Glu Phe 130 135 140 Leu Arg Glu
Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile 145 150 155 160
Ile Val Gly Asp Asp Ser Pro Ile Ala Leu Lys Phe Ala Asn Leu Leu 165
170 175 Val Glu Gly Ser Lys Thr Pro Leu Ala Pro Val Leu Thr Met Gly
Thr 180 185 190 Arg Glu Ala Glu Ala Val Lys Leu Phe Ser Asn Thr Tyr
Leu Ala Met 195 200 205 Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr Phe
Ala Met Ser His Gly 210 215 220 Met Asn Ala Lys Glu Ile Ile Asp Gly
Val Thr Leu Glu Pro Arg Ile 225 230 235 240 Gly Gln Gly Tyr Ser Asn
Pro Ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255 Pro Lys Asp Thr
Lys Gln Leu Leu Ala Asn Phe Glu Gly Val Pro Gln 260 265 270 Asp Ile
Ile Gly Ala Ile Val Glu Ser Asn Glu Thr Arg Lys Glu Val 275 280 285
Ile Val Ser Glu Val Glu Asn Arg Phe Pro Thr Thr Val Gly Val Tyr 290
295 300 Lys Leu Ala Ala Lys Ala Gly Ser Asp Asn Phe Arg Ser Ser Ala
Ile 305 310 315 320 Val Asp Ile Met Glu Arg Leu Ala Asn Lys Gly Tyr
His Ile Lys Ile 325 330 335 Phe Glu Pro Thr Val Glu Gln Phe Glu Asn
Phe Glu Val Asp Asn Asn 340 345 350 Leu Thr Thr Phe Ala Thr Glu Ser
Asp Val Ile Ile Ala Asn Arg Val 355 360 365 Pro Val Glu His Arg Ile
Leu Phe Gly Lys Lys Leu Ile Thr Arg Asp 370 375 380 Val Tyr Gly Asp
Asn 385 <210> SEQ ID NO 13 <211> LENGTH: 1170
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
sequence encoding a Paramecium bursaria Chlorella Virus protein
having the activity of a UDP-Glc-DH <400> SEQUENCE: 13
atgtctcgca tagctgttgt aggatgtggc tatgtgggaa ctgcatgtgc ggttctactt
60 gctcaaaaga acgaagttat tgtgcttgat attagtgaag accgtgttca
acttattaag 120 aacaagaagt ctcctattga ggataaggaa atcgaagagt
tcttggaaac aaaggatctt 180 aatcttactg cgactacaga taaggttctt
gcctacgaga acgctgagtt tgtgataatc 240 gctacaccaa ccgattacga
cgttgtgact cgatatttca ataccaaatc cgtggaaaac 300 gttataggag
atgttatcaa gaacactcaa acccacccta ctatcgtcat caagtccaca 360
attcccatcg gtttcgttga taaggtcaga gagcagtttg attatcaaaa cattatcttc
420 tcacctgagt tcttaaggga gggtcgtgct ctctacgata atttgtatcc
gtcccgtatt 480 atcgttggcg acgattctcc tatcgctctc aagttcgcaa
atctcttagt tgagggtagt 540 aagacccctt tggctcctgt tttgacaatg
ggaaccagag aagcagaagc tgtcaagcta 600 ttctctaata cctaccttgc
catgagggta gcatacttta acgaacttga tacatttgct 660 atgtcgcatg
gtatgaatgc caaggagatt atagatggtg tcactttaga gcccaggatc 720
ggtcaaggat attctaaccc atcattcggc tatggagctt actgctttcc taaggacact
780 aagcagttgc tggcaaactt cgagggagtt cctcaagaca tcataggcgc
tattgtggag 840 tcaaacgaaa caaggaaaga ggtgatagtt agtgaggtag
agaatcgttt cccaacgaca 900 gtcggtgttt acaaactggc agctaaagct
ggtagcgata acttcaggtc aagtgctatt 960 gtcgacatca tggaacgcct
ggctaacaaa ggttaccaca ttaagatctt tgagccaact 1020 gtagagcagt
tcgaaaattt cgaagttgac aataacttga caacgtttgc tactgagtca 1080
gacgttatta tcgcaaatcg tgtccctgtg gaacatagaa tcctatttgg aaagaagctc
1140 attaccagag atgtttacgg tgataattaa 1170 <210> SEQ ID NO 14
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: synthetic oligonucleotide <400> SEQUENCE: 14
tcgacaggcc tggatcctta attaaactag tctcgaggag ctcggtac 48 <210>
SEQ ID NO 15 <211> LENGTH: 40 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: synthetic oligonucleotide
<400> SEQUENCE: 15 cgagctcctc gagactagtt taattaagga
tccaggcctg 40 <210> SEQ ID NO 16 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: synthetic
oligonucleotide <400> SEQUENCE: 16 aaaaactagt tctacatcgg
cttaggtgta gcaacacg 38 <210> SEQ ID NO 17 <211> LENGTH:
39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: synthetic
oligonucleotide <400> SEQUENCE: 17 aaaagatatc tgttgttgga
ttctactact atgcttcaa 39
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 17 <210>
SEQ ID NO 1 <211> LENGTH: 1707 <212> TYPE: DNA
<213> ORGANISM: Paramecium bursaria Chlorella Virus 1
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1707) <300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: PB42580 <309> DATABASE
ENTRY DATE: 1995-12-24 <313> RELEVANT RESIDUES IN SEQ ID NO:
(50903)..(52609) <400> SEQUENCE: 1 atg ggt aaa aat ata atc
ata atg gtt tcg tgg tac acc atc ata act 48 Met Gly Lys Asn Ile Ile
Ile Met Val Ser Trp Tyr Thr Ile Ile Thr 1 5 10 15 tca aat cta atc
gcg gtt gga gga gcc tct cta atc ttg gct ccg gca 96 Ser Asn Leu Ile
Ala Val Gly Gly Ala Ser Leu Ile Leu Ala Pro Ala 20 25 30 att act
ggg tat gtt cta cat tgg aat att gct ctc tcg aca atc tgg 144 Ile Thr
Gly Tyr Val Leu His Trp Asn Ile Ala Leu Ser Thr Ile Trp 35 40 45
gga gta tca gct tat ggt att ttc gtt ttt ggg ttt ttc ctt gca caa 192
Gly Val Ser Ala Tyr Gly Ile Phe Val Phe Gly Phe Phe Leu Ala Gln 50
55 60 gtt tta ttt tca gaa ctg aac agg aaa cgt ctt cgc aag tgg att
tct 240 Val Leu Phe Ser Glu Leu Asn Arg Lys Arg Leu Arg Lys Trp Ile
Ser 65 70 75 80 ctc aga cct aag ggt tgg aat gat gtt cgt ttg gct gtg
atc att gct 288 Leu Arg Pro Lys Gly Trp Asn Asp Val Arg Leu Ala Val
Ile Ile Ala 85 90 95 gga tat cgc gag gat cct tat atg ttc cag aag
tgc ctc gag tct gta 336 Gly Tyr Arg Glu Asp Pro Tyr Met Phe Gln Lys
Cys Leu Glu Ser Val 100 105 110 cgt gac tct gat tat ggc aac gtt gcc
cgt ctg att tgt gtg att gac 384 Arg Asp Ser Asp Tyr Gly Asn Val Ala
Arg Leu Ile Cys Val Ile Asp 115 120 125 ggt gat gag gac gat gat atg
agg atg gct gcc gtt tac aag gcg atc 432 Gly Asp Glu Asp Asp Asp Met
Arg Met Ala Ala Val Tyr Lys Ala Ile 130 135 140 tac aat gat aat atc
aag aag ccc gag ttt gtt ctg tgt gag tca gac 480 Tyr Asn Asp Asn Ile
Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp 145 150 155 160 gac aag
gaa ggt gaa cgc atc gac tct gat ttc tct cgc gac att tgt 528 Asp Lys
Glu Gly Glu Arg Ile Asp Ser Asp Phe Ser Arg Asp Ile Cys 165 170 175
gtc ctc cag cct cat cgt gga aaa cgg gag tgt ctt tat act ggg ttt 576
Val Leu Gln Pro His Arg Gly Lys Arg Glu Cys Leu Tyr Thr Gly Phe 180
185 190 caa ctt gca aag atg gac ccc agt gtc aat gct gtc gtt ctg att
gac 624 Gln Leu Ala Lys Met Asp Pro Ser Val Asn Ala Val Val Leu Ile
Asp 195 200 205 agc gat acc gtt ctc gag aag gat gct att ctg gaa gtt
gta tac cca 672 Ser Asp Thr Val Leu Glu Lys Asp Ala Ile Leu Glu Val
Val Tyr Pro 210 215 220 ctt gca tgc gat ccc gag atc caa gcc gtt gca
ggt gag tgt aag att 720 Leu Ala Cys Asp Pro Glu Ile Gln Ala Val Ala
Gly Glu Cys Lys Ile 225 230 235 240 tgg aac aca gac act ctt ttg agt
ctt ctc gtc gct tgg cgg tac tat 768 Trp Asn Thr Asp Thr Leu Leu Ser
Leu Leu Val Ala Trp Arg Tyr Tyr 245 250 255 tct gcg ttt tgt gtg gag
agg agt gcc cag tct ttt ttc agg act gtt 816 Ser Ala Phe Cys Val Glu
Arg Ser Ala Gln Ser Phe Phe Arg Thr Val 260 265 270 cag tgc gtt ggg
ggg cca ctg ggt gcc tac aag att gat atc att aag 864 Gln Cys Val Gly
Gly Pro Leu Gly Ala Tyr Lys Ile Asp Ile Ile Lys 275 280 285 gag att
aag gac ccc tgg att tcc cag cgc ttt ctt ggt cag aag tgt 912 Glu Ile
Lys Asp Pro Trp Ile Ser Gln Arg Phe Leu Gly Gln Lys Cys 290 295 300
act tac ggt gac gac cgc cgg cta acc aac gag atc ttg atg cgt ggt 960
Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile Leu Met Arg Gly 305
310 315 320 aaa aag gtt gtg ttc act cca ttt gct gtt ggt tgg tct gac
agt ccg 1008 Lys Lys Val Val Phe Thr Pro Phe Ala Val Gly Trp Ser
Asp Ser Pro 325 330 335 acc aat gtg ttt cgg tac atc gtt cag cag acc
cgc tgg agt aag tcg 1056 Thr Asn Val Phe Arg Tyr Ile Val Gln Gln
Thr Arg Trp Ser Lys Ser 340 345 350 tgg tgc cgc gaa att tgg tac acc
ctc ttc gcc gcg tgg aag cac ggt 1104 Trp Cys Arg Glu Ile Trp Tyr
Thr Leu Phe Ala Ala Trp Lys His Gly 355 360 365 ttg tct gga att tgg
ctg gcc ttt gaa tgt ttg tat caa att aca tac 1152 Leu Ser Gly Ile
Trp Leu Ala Phe Glu Cys Leu Tyr Gln Ile Thr Tyr 370 375 380 ttc ttc
ctc gtg att tac ctc ttt tct cgc cta gcc gtt gag gcc gac 1200 Phe
Phe Leu Val Ile Tyr Leu Phe Ser Arg Leu Ala Val Glu Ala Asp 385 390
395 400 cct cgc gcc cag aca gcc acg gtg att gtg agc acc acg gtt gca
ttg 1248 Pro Arg Ala Gln Thr Ala Thr Val Ile Val Ser Thr Thr Val
Ala Leu 405 410 415 att aag tgt ggg tat ttt tca ttc cga gcc aag gat
att cgg gcg ttt 1296 Ile Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys
Asp Ile Arg Ala Phe 420 425 430 tac ttt gtg ctt tat aca ttt gtt tac
ttt ttc tgt atg att ccg gcc 1344 Tyr Phe Val Leu Tyr Thr Phe Val
Tyr Phe Phe Cys Met Ile Pro Ala 435 440 445 agg att act gca atg atg
acg ctt tgg gac att ggc tgg ggt act cgc 1392 Arg Ile Thr Ala Met
Met Thr Leu Trp Asp Ile Gly Trp Gly Thr Arg 450 455 460 ggt gga aac
gag aag cct tcc gtt ggc acc cgg gtc gct ctg tgg gca 1440 Gly Gly
Asn Glu Lys Pro Ser Val Gly Thr Arg Val Ala Leu Trp Ala 465 470 475
480 aag caa tat ctc att gca tat atg tgg tgg gcc gcg gtt gtt ggc gct
1488 Lys Gln Tyr Leu Ile Ala Tyr Met Trp Trp Ala Ala Val Val Gly
Ala 485 490 495 gga gtt tac agc atc gtc cat aac tgg atg ttc gat tgg
aat tct ctt 1536 Gly Val Tyr Ser Ile Val His Asn Trp Met Phe Asp
Trp Asn Ser Leu 500 505 510 tct tat cgt ttt gct ttg gtt ggt att tgt
tct tac att gtt ttt att 1584 Ser Tyr Arg Phe Ala Leu Val Gly Ile
Cys Ser Tyr Ile Val Phe Ile 515 520 525 gtt att gtg ctg gtg gtt tat
ttc acc ggc aaa att acg act tgg aat 1632 Val Ile Val Leu Val Val
Tyr Phe Thr Gly Lys Ile Thr Thr Trp Asn 530 535 540 ttc acg aag ctt
cag aag gag cta atc gag gat cgc gtt ctg tac gat 1680 Phe Thr Lys
Leu Gln Lys Glu Leu Ile Glu Asp Arg Val Leu Tyr Asp 545 550 555 560
gca act acc aat gct cag tct gtg tga 1707 Ala Thr Thr Asn Ala Gln
Ser Val 565 <210> SEQ ID NO 2 <211> LENGTH: 568
<212> TYPE: PRT <213> ORGANISM: Paramecium bursaria
Chlorella Virus 1 <400> SEQUENCE: 2 Met Gly Lys Asn Ile Ile
Ile Met Val Ser Trp Tyr Thr Ile Ile Thr 1 5 10 15 Ser Asn Leu Ile
Ala Val Gly Gly Ala Ser Leu Ile Leu Ala Pro Ala 20 25 30 Ile Thr
Gly Tyr Val Leu His Trp Asn Ile Ala Leu Ser Thr Ile Trp 35 40 45
Gly Val Ser Ala Tyr Gly Ile Phe Val Phe Gly Phe Phe Leu Ala Gln 50
55 60 Val Leu Phe Ser Glu Leu Asn Arg Lys Arg Leu Arg Lys Trp Ile
Ser 65 70 75 80 Leu Arg Pro Lys Gly Trp Asn Asp Val Arg Leu Ala Val
Ile Ile Ala 85 90 95 Gly Tyr Arg Glu Asp Pro Tyr Met Phe Gln Lys
Cys Leu Glu Ser Val 100 105 110 Arg Asp Ser Asp Tyr Gly Asn Val Ala
Arg Leu Ile Cys Val Ile Asp 115 120 125 Gly Asp Glu Asp Asp Asp Met
Arg Met Ala Ala Val Tyr Lys Ala Ile 130 135 140 Tyr Asn Asp Asn Ile
Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp 145 150 155 160 Asp Lys
Glu Gly Glu Arg Ile Asp Ser Asp Phe Ser Arg Asp Ile Cys 165 170 175
Val Leu Gln Pro His Arg Gly Lys Arg Glu Cys Leu Tyr Thr Gly Phe 180
185 190 Gln Leu Ala Lys Met Asp Pro Ser Val Asn Ala Val Val Leu Ile
Asp 195 200 205 Ser Asp Thr Val Leu Glu Lys Asp Ala Ile Leu Glu Val
Val Tyr Pro 210 215 220 Leu Ala Cys Asp Pro Glu Ile Gln Ala Val Ala
Gly Glu Cys Lys Ile 225 230 235 240 Trp Asn Thr Asp Thr Leu Leu Ser
Leu Leu Val Ala Trp Arg Tyr Tyr 245 250 255 Ser Ala Phe Cys Val Glu
Arg Ser Ala Gln Ser Phe Phe Arg Thr Val 260 265 270 Gln Cys Val Gly
Gly Pro Leu Gly Ala Tyr Lys Ile Asp Ile Ile Lys 275 280 285 Glu Ile
Lys Asp Pro Trp Ile Ser Gln Arg Phe Leu Gly Gln Lys Cys 290 295 300
Thr Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile Leu Met Arg Gly 305
310 315 320 Lys Lys Val Val Phe Thr Pro Phe Ala Val Gly Trp Ser Asp
Ser Pro 325 330 335 Thr Asn Val Phe Arg Tyr Ile Val Gln Gln Thr Arg
Trp Ser Lys Ser 340 345 350 Trp Cys Arg Glu Ile Trp Tyr Thr Leu Phe
Ala Ala Trp Lys His Gly 355 360 365 Leu Ser Gly Ile Trp Leu Ala Phe
Glu Cys Leu Tyr Gln Ile Thr Tyr 370 375 380 Phe Phe Leu Val Ile Tyr
Leu Phe Ser Arg Leu Ala Val Glu Ala Asp 385 390 395 400 Pro Arg Ala
Gln Thr Ala Thr Val Ile Val Ser Thr Thr Val Ala Leu 405 410 415
Ile Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys Asp Ile Arg Ala Phe 420
425 430 Tyr Phe Val Leu Tyr Thr Phe Val Tyr Phe Phe Cys Met Ile Pro
Ala 435 440 445 Arg Ile Thr Ala Met Met Thr Leu Trp Asp Ile Gly Trp
Gly Thr Arg 450 455 460 Gly Gly Asn Glu Lys Pro Ser Val Gly Thr Arg
Val Ala Leu Trp Ala 465 470 475 480 Lys Gln Tyr Leu Ile Ala Tyr Met
Trp Trp Ala Ala Val Val Gly Ala 485 490 495 Gly Val Tyr Ser Ile Val
His Asn Trp Met Phe Asp Trp Asn Ser Leu 500 505 510 Ser Tyr Arg Phe
Ala Leu Val Gly Ile Cys Ser Tyr Ile Val Phe Ile 515 520 525 Val Ile
Val Leu Val Val Tyr Phe Thr Gly Lys Ile Thr Thr Trp Asn 530 535 540
Phe Thr Lys Leu Gln Lys Glu Leu Ile Glu Asp Arg Val Leu Tyr Asp 545
550 555 560 Ala Thr Thr Asn Ala Gln Ser Val 565 <210> SEQ ID
NO 3 <211> LENGTH: 1707 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic sequence encoding Paramecium bursaria
Chlorella Virus Hyaluronansynthase protein <400> SEQUENCE: 3
atgggtaaga acattatcat tatggtgtcc tggtacacaa ttattacaag taatctcatc
60 gcagttggtg gtgcatctct tattctcgct ccagctatca ctggatatgt
tcttcactgg 120 aacatcgccc tctcaactat ttggggagtt tccgcatatg
gtatttttgt tttcgggttc 180 tttttggctc aggttctgtt ctcagagctc
aatcgtaaga gactcaggaa gtggattagc 240 cttagaccaa aggggtggaa
tgacgttcgt ctcgctgtca ttatcgctgg ctaccgtgaa 300 gatccttaca
tgtttcaaaa gtgcttggaa tcagttaggg atagtgatta tggcaacgtc 360
gctagactga tctgtgtgat tgatggagat gaggacgacg atatgaggat ggcagctgtt
420 tataaggcta tctataatga taacattaag aagcctgaat ttgttctttg
cgagtctgat 480 gacaaggaag gagaacggat tgattcagat ttctcacgtg
atatctgcgt tctccaacct 540 catcgtggga agcgtgaatg tctttataca
ggtttccaac tcgccaaaat ggacccatca 600 gtgaacgctg tggttcttat
cgatagtgat actgtgctgg agaaagatgc tatcttggag 660 gttgtttacc
ctcttgcctg tgatcctgaa attcaagctg tggctggaga gtgcaagatc 720
tggaacacag atactcttct ttctctgctt gtcgcatgga gatattactc cgcattctgt
780 gtggagagga gcgctcaatc ctttttccgt accgttcaat gcgttggtgg
tcctttggga 840 gcttacaaaa ttgatatcat caaggagatt aaggacccat
ggattagtca aaggtttctt 900 ggtcagaagt gcacttatgg cgatgatcgt
agattgacta acgaaatcct tatgaggggc 960 aagaaagtcg tttttactcc
atttgctgtc ggatggtctg attcacctac aaatgttttc 1020 cgttatattg
tgcaacaaac acgttggagt aagagctggt gtagggagat ctggtacact 1080
ttgttcgctg cttggaagca cgggcttagc ggaatttggc ttgcttttga atgcctttac
1140 cagattacat actttttctt ggtgatctat ttgttttcac gtcttgccgt
cgaggctgac 1200 cctagagcac agactgcaac tgtgattgtt tctactacag
tcgcacttat taagtgtggc 1260 tatttcagtt ttagagcaaa agatattaga
gccttctatt ttgttttgta cacatttgtt 1320 tatttctttt gcatgattcc
agctcgtatt accgctatga tgaccttgtg ggacatcgga 1380 tggggaacta
gaggtggtaa cgaaaagcct tctgtgggaa caagggtggc cctttgggca 1440
aaacaatatc tcatcgccta catgtggtgg gccgctgtcg ttggtgccgg agtgtactca
1500 atcgttcata actggatgtt tgactggaac tctttgagct atcgtttcgc
tcttgtgggt 1560 atttgttctt acattgtttt catcgtgatt gtgctcgttg
tgtatttcac tggtaaaatc 1620 acaacctgga atttcactaa acttcaaaag
gaattgattg aagacagggt tctgtatgat 1680 gctactacca acgcccagtc agtttaa
1707 <210> SEQ ID NO 4 <211> LENGTH: 2298 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (150)..(2192)
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: BC050762.1 <309> DATABASE ENTRY DATE: 2005-03-08
<313> RELEVANT RESIDUES IN SEQ ID NO: (150)..(2195)
<400> SEQUENCE: 4 gagagcgaag cgagcgctga gtcggactgt cgggtctgag
ctgtcgcatc ccagagtcct 60 ctcattgcca ccaccccggc ccgagctcac
cctcgcttct gaagctctcc gcgcgcccga 120 cagctcagcc ctcgcccgtg
accaacatc atg tgc ggt ata ttt gct tat tta 173 Met Cys Gly Ile Phe
Ala Tyr Leu 1 5 aat tac cat gtt cct cga aca aga cga gaa atc ttg gag
aca cta atc 221 Asn Tyr His Val Pro Arg Thr Arg Arg Glu Ile Leu Glu
Thr Leu Ile 10 15 20 aaa ggc ctt cag aga ctg gaa tac aga gga tat
gat tct gct ggt gtg 269 Lys Gly Leu Gln Arg Leu Glu Tyr Arg Gly Tyr
Asp Ser Ala Gly Val 25 30 35 40 gga ctt gac gga ggc aat gac aaa gac
tgg gaa gcc aac gcc tgc aaa 317 Gly Leu Asp Gly Gly Asn Asp Lys Asp
Trp Glu Ala Asn Ala Cys Lys 45 50 55 atc cag ctc att aag aag aaa
gga aaa gtt aag gca ctg gat gaa gaa 365 Ile Gln Leu Ile Lys Lys Lys
Gly Lys Val Lys Ala Leu Asp Glu Glu 60 65 70 gtt cac aaa caa caa
gat atg gac ttg gat ata gaa ttt gat gtg cat 413 Val His Lys Gln Gln
Asp Met Asp Leu Asp Ile Glu Phe Asp Val His 75 80 85 ctt gga ata
gct cat acc cgt tgg gcg aca cat gga gaa ccc aat cct 461 Leu Gly Ile
Ala His Thr Arg Trp Ala Thr His Gly Glu Pro Asn Pro 90 95 100 gtc
aat agt cac ccc cag cgc tct gat aaa aat aat gaa ttc att gtt 509 Val
Asn Ser His Pro Gln Arg Ser Asp Lys Asn Asn Glu Phe Ile Val 105 110
115 120 att cat aat gga atc atc acc aac tac aaa gac ttg aaa aag ttt
ctg 557 Ile His Asn Gly Ile Ile Thr Asn Tyr Lys Asp Leu Lys Lys Phe
Leu 125 130 135 gaa agc aaa ggc tat gac ttt gaa tct gaa aca gac aca
gaa acc att 605 Glu Ser Lys Gly Tyr Asp Phe Glu Ser Glu Thr Asp Thr
Glu Thr Ile 140 145 150 gcc aag ctc gtc aag tac atg tat gac aac tgg
gag agc cag gac gtc 653 Ala Lys Leu Val Lys Tyr Met Tyr Asp Asn Trp
Glu Ser Gln Asp Val 155 160 165 agt ttt acc acc ttg gtg gag aga gtt
atc caa caa ttg gaa ggc gcc 701 Ser Phe Thr Thr Leu Val Glu Arg Val
Ile Gln Gln Leu Glu Gly Ala 170 175 180 ttt gct ctt gtg ttt aaa agt
gtc cat ttt ccc ggg caa gca gtt ggc 749 Phe Ala Leu Val Phe Lys Ser
Val His Phe Pro Gly Gln Ala Val Gly 185 190 195 200 aca agg cga ggt
agc cct ctc ttg att ggt gtg cgg agt gaa cat aag 797 Thr Arg Arg Gly
Ser Pro Leu Leu Ile Gly Val Arg Ser Glu His Lys 205 210 215 ctt tct
aca gat cac att ccg att ctg tac aga aca ggc aaa gac aag 845 Leu Ser
Thr Asp His Ile Pro Ile Leu Tyr Arg Thr Gly Lys Asp Lys 220 225 230
aaa gga agc tgc ggt ctt tcc cgt gtg gac agc acg aca tgc ctg ttc 893
Lys Gly Ser Cys Gly Leu Ser Arg Val Asp Ser Thr Thr Cys Leu Phe 235
240 245 cct gtt gag gaa aag gca gtt gaa tat tac ttt gct tct gat gca
agt 941 Pro Val Glu Glu Lys Ala Val Glu Tyr Tyr Phe Ala Ser Asp Ala
Ser 250 255 260 gcc gtg ata gag cac acc aat cgt gtc atc ttt ctg gaa
gat gat gat 989 Ala Val Ile Glu His Thr Asn Arg Val Ile Phe Leu Glu
Asp Asp Asp 265 270 275 280 gtt gca gca gtg gtg gat ggc cgt ctc tct
atc cac cga att aaa cga 1037 Val Ala Ala Val Val Asp Gly Arg Leu
Ser Ile His Arg Ile Lys Arg 285 290 295 act gca gga gac cat cct ggc
cga gct gtg caa act ctc cag atg gag 1085 Thr Ala Gly Asp His Pro
Gly Arg Ala Val Gln Thr Leu Gln Met Glu 300 305 310 ctc cag cag atc
atg aag ggc aac ttt agt tca ttt atg cag aag gaa 1133 Leu Gln Gln
Ile Met Lys Gly Asn Phe Ser Ser Phe Met Gln Lys Glu 315 320 325 att
ttt gag cag cca gaa tct gtt gtg aac aca atg aga gga aga gtc 1181
Ile Phe Glu Gln Pro Glu Ser Val Val Asn Thr Met Arg Gly Arg Val 330
335 340 aat ttt gat gac tac act gtg aat ttg gga ggt ttg aaa gat cac
att 1229 Asn Phe Asp Asp Tyr Thr Val Asn Leu Gly Gly Leu Lys Asp
His Ile 345 350 355 360 aag gag atc cag cgg tgt cgg cgg ttg att ctt
att gct tgt ggc aca 1277 Lys Glu Ile Gln Arg Cys Arg Arg Leu Ile
Leu Ile Ala Cys Gly Thr 365 370 375 agt tac cac gct ggt gtg gca acc
cgt cag gtc ctg gag gag ctg acc 1325 Ser Tyr His Ala Gly Val Ala
Thr Arg Gln Val Leu Glu Glu Leu Thr 380 385 390 gag ctg ccc gtg atg
gtg gag ctt gcc agt gac ttc ttg gat aga aac 1373 Glu Leu Pro Val
Met Val Glu Leu Ala Ser Asp Phe Leu Asp Arg Asn 395 400 405 act cca
gtc ttt cga gat gat gtt tgc ttt ttc att agt caa tca ggc 1421 Thr
Pro Val Phe Arg Asp Asp Val Cys Phe Phe Ile Ser Gln Ser Gly 410 415
420 gag aca gct gac acc ctg atg gga ctt cgt tac tgt aag gag aga gga
1469 Glu Thr Ala Asp Thr Leu Met Gly Leu Arg Tyr Cys Lys Glu Arg
Gly 425 430 435 440 gcc tta act gtg ggg atc aca aat aca gtc ggc agt
tct ata tca agg 1517 Ala Leu Thr Val Gly Ile Thr Asn Thr Val Gly
Ser Ser Ile Ser Arg 445 450 455 gag aca gat tgc ggg gtt cat att aat
gct ggt cct gag att ggc gtg 1565 Glu Thr Asp Cys Gly Val His Ile
Asn Ala Gly Pro Glu Ile Gly Val 460 465 470 gcc agt aca aag gca tac
acc agc cag ttt gtg tcc ctc gtg atg ttt 1613 Ala Ser Thr Lys Ala
Tyr Thr Ser Gln Phe Val Ser Leu Val Met Phe 475 480 485 gct ctc atg
atg tgt gat gac agg atc tcc atg caa gag aga cgc aaa 1661 Ala Leu
Met Met Cys Asp Asp Arg Ile Ser Met Gln Glu Arg Arg Lys 490 495 500
gag atc atg ctc gga ctg aag cga ctg ccg gac ttg att aag gaa gtg
1709 Glu Ile Met Leu Gly Leu Lys Arg Leu Pro Asp Leu Ile Lys Glu
Val 505 510 515 520
ctg agc atg gat gat gaa atc cag aag ctg gcg acg gag ctt tac cac
1757 Leu Ser Met Asp Asp Glu Ile Gln Lys Leu Ala Thr Glu Leu Tyr
His 525 530 535 cag aag tcg gtc ctg ata atg ggg cgg ggc tac cat tat
gct aca tgc 1805 Gln Lys Ser Val Leu Ile Met Gly Arg Gly Tyr His
Tyr Ala Thr Cys 540 545 550 ctt gaa ggg gct ctg aaa atc aag gag att
act tat atg cat tcg gaa 1853 Leu Glu Gly Ala Leu Lys Ile Lys Glu
Ile Thr Tyr Met His Ser Glu 555 560 565 ggc atc ctt gct ggt gag ctc
aag cac ggc cct ctg gcc ttg gtg gac 1901 Gly Ile Leu Ala Gly Glu
Leu Lys His Gly Pro Leu Ala Leu Val Asp 570 575 580 aag ttg atg cct
gtc atc atg atc atc atg cga gac cac act tat gcc 1949 Lys Leu Met
Pro Val Ile Met Ile Ile Met Arg Asp His Thr Tyr Ala 585 590 595 600
aag tgc cag aac gct ctt cag cag gtg gtt gca cgg cag ggg cgt cca
1997 Lys Cys Gln Asn Ala Leu Gln Gln Val Val Ala Arg Gln Gly Arg
Pro 605 610 615 gtc gtg atc tgt gat aag gag gat act gag acc att aag
aat aca aaa 2045 Val Val Ile Cys Asp Lys Glu Asp Thr Glu Thr Ile
Lys Asn Thr Lys 620 625 630 agg aca atc aag gtg ccc cac tca gtg gac
tgc ttg cag ggc att ctc 2093 Arg Thr Ile Lys Val Pro His Ser Val
Asp Cys Leu Gln Gly Ile Leu 635 640 645 agt gtg att ccc ctg cag ctg
ctg gct ttc cac ctg gct gtg ctg aga 2141 Ser Val Ile Pro Leu Gln
Leu Leu Ala Phe His Leu Ala Val Leu Arg 650 655 660 ggc tac gat gtt
gat ttt cca cgg aat ctt gcc aaa tct gta aca gta 2189 Gly Tyr Asp
Val Asp Phe Pro Arg Asn Leu Ala Lys Ser Val Thr Val 665 670 675 680
gag taacagacac ctgaaactta agacagttaa gcaacacgag ataccttttg 2242 Glu
tatttaaatt tttgatttaa actatcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2298
<210> SEQ ID NO 5 <211> LENGTH: 681 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 5 Met
Cys Gly Ile Phe Ala Tyr Leu Asn Tyr His Val Pro Arg Thr Arg 1 5 10
15 Arg Glu Ile Leu Glu Thr Leu Ile Lys Gly Leu Gln Arg Leu Glu Tyr
20 25 30 Arg Gly Tyr Asp Ser Ala Gly Val Gly Leu Asp Gly Gly Asn
Asp Lys 35 40 45 Asp Trp Glu Ala Asn Ala Cys Lys Ile Gln Leu Ile
Lys Lys Lys Gly 50 55 60 Lys Val Lys Ala Leu Asp Glu Glu Val His
Lys Gln Gln Asp Met Asp 65 70 75 80 Leu Asp Ile Glu Phe Asp Val His
Leu Gly Ile Ala His Thr Arg Trp 85 90 95 Ala Thr His Gly Glu Pro
Asn Pro Val Asn Ser His Pro Gln Arg Ser 100 105 110 Asp Lys Asn Asn
Glu Phe Ile Val Ile His Asn Gly Ile Ile Thr Asn 115 120 125 Tyr Lys
Asp Leu Lys Lys Phe Leu Glu Ser Lys Gly Tyr Asp Phe Glu 130 135 140
Ser Glu Thr Asp Thr Glu Thr Ile Ala Lys Leu Val Lys Tyr Met Tyr 145
150 155 160 Asp Asn Trp Glu Ser Gln Asp Val Ser Phe Thr Thr Leu Val
Glu Arg 165 170 175 Val Ile Gln Gln Leu Glu Gly Ala Phe Ala Leu Val
Phe Lys Ser Val 180 185 190 His Phe Pro Gly Gln Ala Val Gly Thr Arg
Arg Gly Ser Pro Leu Leu 195 200 205 Ile Gly Val Arg Ser Glu His Lys
Leu Ser Thr Asp His Ile Pro Ile 210 215 220 Leu Tyr Arg Thr Gly Lys
Asp Lys Lys Gly Ser Cys Gly Leu Ser Arg 225 230 235 240 Val Asp Ser
Thr Thr Cys Leu Phe Pro Val Glu Glu Lys Ala Val Glu 245 250 255 Tyr
Tyr Phe Ala Ser Asp Ala Ser Ala Val Ile Glu His Thr Asn Arg 260 265
270 Val Ile Phe Leu Glu Asp Asp Asp Val Ala Ala Val Val Asp Gly Arg
275 280 285 Leu Ser Ile His Arg Ile Lys Arg Thr Ala Gly Asp His Pro
Gly Arg 290 295 300 Ala Val Gln Thr Leu Gln Met Glu Leu Gln Gln Ile
Met Lys Gly Asn 305 310 315 320 Phe Ser Ser Phe Met Gln Lys Glu Ile
Phe Glu Gln Pro Glu Ser Val 325 330 335 Val Asn Thr Met Arg Gly Arg
Val Asn Phe Asp Asp Tyr Thr Val Asn 340 345 350 Leu Gly Gly Leu Lys
Asp His Ile Lys Glu Ile Gln Arg Cys Arg Arg 355 360 365 Leu Ile Leu
Ile Ala Cys Gly Thr Ser Tyr His Ala Gly Val Ala Thr 370 375 380 Arg
Gln Val Leu Glu Glu Leu Thr Glu Leu Pro Val Met Val Glu Leu 385 390
395 400 Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp Asp
Val 405 410 415 Cys Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr
Leu Met Gly 420 425 430 Leu Arg Tyr Cys Lys Glu Arg Gly Ala Leu Thr
Val Gly Ile Thr Asn 435 440 445 Thr Val Gly Ser Ser Ile Ser Arg Glu
Thr Asp Cys Gly Val His Ile 450 455 460 Asn Ala Gly Pro Glu Ile Gly
Val Ala Ser Thr Lys Ala Tyr Thr Ser 465 470 475 480 Gln Phe Val Ser
Leu Val Met Phe Ala Leu Met Met Cys Asp Asp Arg 485 490 495 Ile Ser
Met Gln Glu Arg Arg Lys Glu Ile Met Leu Gly Leu Lys Arg 500 505 510
Leu Pro Asp Leu Ile Lys Glu Val Leu Ser Met Asp Asp Glu Ile Gln 515
520 525 Lys Leu Ala Thr Glu Leu Tyr His Gln Lys Ser Val Leu Ile Met
Gly 530 535 540 Arg Gly Tyr His Tyr Ala Thr Cys Leu Glu Gly Ala Leu
Lys Ile Lys 545 550 555 560 Glu Ile Thr Tyr Met His Ser Glu Gly Ile
Leu Ala Gly Glu Leu Lys 565 570 575 His Gly Pro Leu Ala Leu Val Asp
Lys Leu Met Pro Val Ile Met Ile 580 585 590 Ile Met Arg Asp His Thr
Tyr Ala Lys Cys Gln Asn Ala Leu Gln Gln 595 600 605 Val Val Ala Arg
Gln Gly Arg Pro Val Val Ile Cys Asp Lys Glu Asp 610 615 620 Thr Glu
Thr Ile Lys Asn Thr Lys Arg Thr Ile Lys Val Pro His Ser 625 630 635
640 Val Asp Cys Leu Gln Gly Ile Leu Ser Val Ile Pro Leu Gln Leu Leu
645 650 655 Ala Phe His Leu Ala Val Leu Arg Gly Tyr Asp Val Asp Phe
Pro Arg 660 665 670 Asn Leu Ala Lys Ser Val Thr Val Glu 675 680
<210> SEQ ID NO 6 <211> LENGTH: 2049 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(2046)
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: BC031928.1 <309> DATABASE ENTRY DATE: 2003-10-07
<313> RELEVANT RESIDUES IN SEQ ID NO: (51)..(299) <400>
SEQUENCE: 6 atg tgc gga atc ttt gcc tac atg aat tac aga gtt ccc aag
aca agg 48 Met Cys Gly Ile Phe Ala Tyr Met Asn Tyr Arg Val Pro Lys
Thr Arg 1 5 10 15 aaa gag att ttc gaa acc ctt atc agg ggt ctg cag
cgg ctg gag tac 96 Lys Glu Ile Phe Glu Thr Leu Ile Arg Gly Leu Gln
Arg Leu Glu Tyr 20 25 30 cgg ggc tat gac tct gcg ggg gtt gcc att
gat ggg aat aac cac gaa 144 Arg Gly Tyr Asp Ser Ala Gly Val Ala Ile
Asp Gly Asn Asn His Glu 35 40 45 gtc aaa gaa aga cac atc cat ctt
gtg aag aaa agg ggg aaa gta aag 192 Val Lys Glu Arg His Ile His Leu
Val Lys Lys Arg Gly Lys Val Lys 50 55 60 gct ctg gat gaa gaa ctt
tac aag caa gat agc atg gac ttg aag gtg 240 Ala Leu Asp Glu Glu Leu
Tyr Lys Gln Asp Ser Met Asp Leu Lys Val 65 70 75 80 gag ttt gag aca
cac ttc ggc att gcc cac aca cgt tgg gcc acc cac 288 Glu Phe Glu Thr
His Phe Gly Ile Ala His Thr Arg Trp Ala Thr His 85 90 95 ggg gtt
ccc aat gct gtc aac agt cac ccg cag cgt tcg gac aaa gac 336 Gly Val
Pro Asn Ala Val Asn Ser His Pro Gln Arg Ser Asp Lys Asp 100 105 110
aat gaa ttt gtt gtc atc cac aac ggg atc atc act aat tac aag gat 384
Asn Glu Phe Val Val Ile His Asn Gly Ile Ile Thr Asn Tyr Lys Asp 115
120 125 cta agg aag ttt ctg gaa agc aaa ggc tac gag ttt gag tca gaa
aca 432 Leu Arg Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe Glu Ser Glu
Thr 130 135 140 gac acg gag acc atc gcc aag ctg att aaa tat gta ttt
gac aac aga 480 Asp Thr Glu Thr Ile Ala Lys Leu Ile Lys Tyr Val Phe
Asp Asn Arg 145 150 155 160 gag act gag gac ata acg ttt tcc aca ttg
gtc gaa aga gtc att cag 528 Glu Thr Glu Asp Ile Thr Phe Ser Thr Leu
Val Glu Arg Val Ile Gln 165 170 175 cag ttg gaa ggc gcc ttt gca ctg
gtt ttc aag agt att cac tac ccg 576 Gln Leu Glu Gly Ala Phe Ala Leu
Val Phe Lys Ser Ile His Tyr Pro 180 185 190 gga gaa gct gtc gcc acg
agg aga ggc agc ccc ttg ctc atc ggg gta 624 Gly Glu Ala Val Ala Thr
Arg Arg Gly Ser Pro Leu Leu Ile Gly Val 195 200 205 cga agc aaa tac
aaa ctc tcc aca gag cag atc ccc gtc tta tat ccg 672 Arg Ser Lys Tyr
Lys Leu Ser Thr Glu Gln Ile Pro Val Leu Tyr Pro
210 215 220 aca tgc aat atc gag aat gtg aag aat atc tgc aag act agg
atg aag 720 Thr Cys Asn Ile Glu Asn Val Lys Asn Ile Cys Lys Thr Arg
Met Lys 225 230 235 240 aga ctg gac agc tcc acc tgc ctg cac gct gtg
ggc gat aaa gct gtg 768 Arg Leu Asp Ser Ser Thr Cys Leu His Ala Val
Gly Asp Lys Ala Val 245 250 255 gaa ttc ttc ttt gct tct gat gca agt
gcc atc ata gaa cac acc aac 816 Glu Phe Phe Phe Ala Ser Asp Ala Ser
Ala Ile Ile Glu His Thr Asn 260 265 270 cgg gtc atc ttc tta gaa gat
gat gat atc gct gca gtg gct gat ggg 864 Arg Val Ile Phe Leu Glu Asp
Asp Asp Ile Ala Ala Val Ala Asp Gly 275 280 285 aaa ctc tcc att cac
cga gtc aag cgc tca gct act gat gac ccc tcc 912 Lys Leu Ser Ile His
Arg Val Lys Arg Ser Ala Thr Asp Asp Pro Ser 290 295 300 cga gcc atc
cag acc ttg cag atg gaa ctg cag caa ata atg aaa ggt 960 Arg Ala Ile
Gln Thr Leu Gln Met Glu Leu Gln Gln Ile Met Lys Gly 305 310 315 320
aac ttc agc gca ttt atg cag aag gag atc ttc gag cag cca gaa tca
1008 Asn Phe Ser Ala Phe Met Gln Lys Glu Ile Phe Glu Gln Pro Glu
Ser 325 330 335 gtt ttt aat acc atg aga ggt cgg gtg aat ttt gag acc
aac aca gtg 1056 Val Phe Asn Thr Met Arg Gly Arg Val Asn Phe Glu
Thr Asn Thr Val 340 345 350 ctc ctg ggt ggc ttg aag gac cat ttg aaa
gag atc cga cga tgc cga 1104 Leu Leu Gly Gly Leu Lys Asp His Leu
Lys Glu Ile Arg Arg Cys Arg 355 360 365 agg ctc att gtg att ggc tgt
gga acc agc tac cat gcc gct gtg gct 1152 Arg Leu Ile Val Ile Gly
Cys Gly Thr Ser Tyr His Ala Ala Val Ala 370 375 380 aca cgg caa gtc
tta gag gaa ctg acc gag ctg cct gtg atg gtt gaa 1200 Thr Arg Gln
Val Leu Glu Glu Leu Thr Glu Leu Pro Val Met Val Glu 385 390 395 400
ctt gcc agt gac ttt ctg gac agg aac aca cct gtg ttc agg gat gac
1248 Leu Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp
Asp 405 410 415 gtt tgc ttt ttc ata agc caa tca ggt gag act gca gac
acg ctc ctg 1296 Val Cys Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala
Asp Thr Leu Leu 420 425 430 gcg ctg cga tac tgt aag gat cga ggt gcg
ctg acc gtg ggc atc acc 1344 Ala Leu Arg Tyr Cys Lys Asp Arg Gly
Ala Leu Thr Val Gly Ile Thr 435 440 445 aac acc gtg ggt agc tcc atc
tcc cgg gag act gac tgt ggc gtc cac 1392 Asn Thr Val Gly Ser Ser
Ile Ser Arg Glu Thr Asp Cys Gly Val His 450 455 460 atc aac gca ggg
ccc gag att ggg gtg gcc agc acc aag gcg tac acc 1440 Ile Asn Ala
Gly Pro Glu Ile Gly Val Ala Ser Thr Lys Ala Tyr Thr 465 470 475 480
agc cag ttc atc tct ctg gtg atg ttt ggt ttg atg atg tct gaa gat
1488 Ser Gln Phe Ile Ser Leu Val Met Phe Gly Leu Met Met Ser Glu
Asp 485 490 495 cga att tct cta cag aac agg aga caa gag atc atc cgt
ggc ctc aga 1536 Arg Ile Ser Leu Gln Asn Arg Arg Gln Glu Ile Ile
Arg Gly Leu Arg 500 505 510 tct tta ccg gag ctg atc aaa gaa gtg ctg
tcc ctg gat gag aag atc 1584 Ser Leu Pro Glu Leu Ile Lys Glu Val
Leu Ser Leu Asp Glu Lys Ile 515 520 525 cat gac ttg gcc ctg gag ctc
tac aca caa agg tct ctc ctc gtg atg 1632 His Asp Leu Ala Leu Glu
Leu Tyr Thr Gln Arg Ser Leu Leu Val Met 530 535 540 gga cgg gga tat
aac tat gcc aca tgt ctg gaa ggt gcc ttg aaa att 1680 Gly Arg Gly
Tyr Asn Tyr Ala Thr Cys Leu Glu Gly Ala Leu Lys Ile 545 550 555 560
aag gag ata acc tac atg cat tca gaa ggt atc cta gcc gga gag ctg
1728 Lys Glu Ile Thr Tyr Met His Ser Glu Gly Ile Leu Ala Gly Glu
Leu 565 570 575 aag cac ggg ccc ctt gct ctc gtc gac aag cag atg cca
gtc atc atg 1776 Lys His Gly Pro Leu Ala Leu Val Asp Lys Gln Met
Pro Val Ile Met 580 585 590 gtc atc atg aag gat cct tgc ttt gcc aag
tgc cag aat gcc ctg cag 1824 Val Ile Met Lys Asp Pro Cys Phe Ala
Lys Cys Gln Asn Ala Leu Gln 595 600 605 cag gtc act gcc cgc cag ggt
cgc cca atc ata ctg tgt tcc aag gat 1872 Gln Val Thr Ala Arg Gln
Gly Arg Pro Ile Ile Leu Cys Ser Lys Asp 610 615 620 gac acc gag agc
tcc aag ttt gca tat aaa acc att gaa ctt ccc cac 1920 Asp Thr Glu
Ser Ser Lys Phe Ala Tyr Lys Thr Ile Glu Leu Pro His 625 630 635 640
aca gtg gac tgt ctc cag ggt atc ctg agc gtg att cca ctc cag ctt
1968 Thr Val Asp Cys Leu Gln Gly Ile Leu Ser Val Ile Pro Leu Gln
Leu 645 650 655 ctg tcc ttc cac ctg gct gtc ctc cga ggt tat gat gtt
gac ttc ccc 2016 Leu Ser Phe His Leu Ala Val Leu Arg Gly Tyr Asp
Val Asp Phe Pro 660 665 670 aga aac cta gcc aag tct gtc act gtg gaa
tga 2049 Arg Asn Leu Ala Lys Ser Val Thr Val Glu 675 680
<210> SEQ ID NO 7 <211> LENGTH: 682 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 7 Met
Cys Gly Ile Phe Ala Tyr Met Asn Tyr Arg Val Pro Lys Thr Arg 1 5 10
15 Lys Glu Ile Phe Glu Thr Leu Ile Arg Gly Leu Gln Arg Leu Glu Tyr
20 25 30 Arg Gly Tyr Asp Ser Ala Gly Val Ala Ile Asp Gly Asn Asn
His Glu 35 40 45 Val Lys Glu Arg His Ile His Leu Val Lys Lys Arg
Gly Lys Val Lys 50 55 60 Ala Leu Asp Glu Glu Leu Tyr Lys Gln Asp
Ser Met Asp Leu Lys Val 65 70 75 80 Glu Phe Glu Thr His Phe Gly Ile
Ala His Thr Arg Trp Ala Thr His 85 90 95 Gly Val Pro Asn Ala Val
Asn Ser His Pro Gln Arg Ser Asp Lys Asp 100 105 110 Asn Glu Phe Val
Val Ile His Asn Gly Ile Ile Thr Asn Tyr Lys Asp 115 120 125 Leu Arg
Lys Phe Leu Glu Ser Lys Gly Tyr Glu Phe Glu Ser Glu Thr 130 135 140
Asp Thr Glu Thr Ile Ala Lys Leu Ile Lys Tyr Val Phe Asp Asn Arg 145
150 155 160 Glu Thr Glu Asp Ile Thr Phe Ser Thr Leu Val Glu Arg Val
Ile Gln 165 170 175 Gln Leu Glu Gly Ala Phe Ala Leu Val Phe Lys Ser
Ile His Tyr Pro 180 185 190 Gly Glu Ala Val Ala Thr Arg Arg Gly Ser
Pro Leu Leu Ile Gly Val 195 200 205 Arg Ser Lys Tyr Lys Leu Ser Thr
Glu Gln Ile Pro Val Leu Tyr Pro 210 215 220 Thr Cys Asn Ile Glu Asn
Val Lys Asn Ile Cys Lys Thr Arg Met Lys 225 230 235 240 Arg Leu Asp
Ser Ser Thr Cys Leu His Ala Val Gly Asp Lys Ala Val 245 250 255 Glu
Phe Phe Phe Ala Ser Asp Ala Ser Ala Ile Ile Glu His Thr Asn 260 265
270 Arg Val Ile Phe Leu Glu Asp Asp Asp Ile Ala Ala Val Ala Asp Gly
275 280 285 Lys Leu Ser Ile His Arg Val Lys Arg Ser Ala Thr Asp Asp
Pro Ser 290 295 300 Arg Ala Ile Gln Thr Leu Gln Met Glu Leu Gln Gln
Ile Met Lys Gly 305 310 315 320 Asn Phe Ser Ala Phe Met Gln Lys Glu
Ile Phe Glu Gln Pro Glu Ser 325 330 335 Val Phe Asn Thr Met Arg Gly
Arg Val Asn Phe Glu Thr Asn Thr Val 340 345 350 Leu Leu Gly Gly Leu
Lys Asp His Leu Lys Glu Ile Arg Arg Cys Arg 355 360 365 Arg Leu Ile
Val Ile Gly Cys Gly Thr Ser Tyr His Ala Ala Val Ala 370 375 380 Thr
Arg Gln Val Leu Glu Glu Leu Thr Glu Leu Pro Val Met Val Glu 385 390
395 400 Leu Ala Ser Asp Phe Leu Asp Arg Asn Thr Pro Val Phe Arg Asp
Asp 405 410 415 Val Cys Phe Phe Ile Ser Gln Ser Gly Glu Thr Ala Asp
Thr Leu Leu 420 425 430 Ala Leu Arg Tyr Cys Lys Asp Arg Gly Ala Leu
Thr Val Gly Ile Thr 435 440 445 Asn Thr Val Gly Ser Ser Ile Ser Arg
Glu Thr Asp Cys Gly Val His 450 455 460 Ile Asn Ala Gly Pro Glu Ile
Gly Val Ala Ser Thr Lys Ala Tyr Thr 465 470 475 480 Ser Gln Phe Ile
Ser Leu Val Met Phe Gly Leu Met Met Ser Glu Asp 485 490 495 Arg Ile
Ser Leu Gln Asn Arg Arg Gln Glu Ile Ile Arg Gly Leu Arg 500 505 510
Ser Leu Pro Glu Leu Ile Lys Glu Val Leu Ser Leu Asp Glu Lys Ile 515
520 525 His Asp Leu Ala Leu Glu Leu Tyr Thr Gln Arg Ser Leu Leu Val
Met 530 535 540 Gly Arg Gly Tyr Asn Tyr Ala Thr Cys Leu Glu Gly Ala
Leu Lys Ile 545 550 555 560 Lys Glu Ile Thr Tyr Met His Ser Glu Gly
Ile Leu Ala Gly Glu Leu 565 570 575 Lys His Gly Pro Leu Ala Leu Val
Asp Lys Gln Met Pro Val Ile Met 580 585 590 Val Ile Met Lys Asp Pro
Cys Phe Ala Lys Cys Gln Asn Ala Leu Gln 595 600 605 Gln Val Thr Ala
Arg Gln Gly Arg Pro Ile Ile Leu Cys Ser Lys Asp 610 615 620 Asp Thr
Glu Ser Ser Lys Phe Ala Tyr Lys Thr Ile Glu Leu Pro His 625 630 635
640 Thr Val Asp Cys Leu Gln Gly Ile Leu Ser Val Ile Pro Leu Gln Leu
645 650 655 Leu Ser Phe His Leu Ala Val Leu Arg Gly Tyr Asp Val Asp
Phe Pro 660 665 670
Arg Asn Leu Ala Lys Ser Val Thr Val Glu 675 680 <210> SEQ ID
NO 8 <211> LENGTH: 1830 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (1)..(1827) <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
U00096.2 <309> DATABASE ENTRY DATE: 2005-09-08 <313>
RELEVANT RESIDUES IN SEQ ID NO: (3909862)..(3911691) <400>
SEQUENCE: 8 atg tgt gga att gtt ggc gcg atc gcg caa cgt gat gta gca
gaa atc 48 Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala
Glu Ile 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga
tat gac tct gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly
Tyr Asp Ser Ala 20 25 30 ggt ctg gcc gtt gtt gat gca gaa ggt cat
atg acc cgc ctg cgt cgc 144 Gly Leu Ala Val Val Asp Ala Glu Gly His
Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gca
cag gca gcg gaa gaa cat cct ctg 192 Leu Gly Lys Val Gln Met Leu Ala
Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att
gct cac act cgc tgg gcg acc cac ggt gaa 240 His Gly Gly Thr Gly Ile
Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 cct tca gaa gtg
aat gcg cat ccg cat gtt tct gaa cac att gtg gtg 288 Pro Ser Glu Val
Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95 gtg cat
aac ggc atc atc gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His
Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110
aaa gcg cgt ggc tat acc ttc gtt tct gaa acc gac acc gaa gtg att 384
Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115
120 125 gcc cat ctg gtg aac tgg gag ctg aaa caa ggc ggg act ctg cgt
gag 432 Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg
Glu 130 135 140 gcc gtt ctg cgt gct atc ccg cag ctg cgt ggt gcg tac
ggt aca gtg 480 Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr
Gly Thr Val 145 150 155 160 atc atg gac tcc cgt cac ccg gat acc ctg
ctg gcg gca cgt tct ggt 528 Ile Met Asp Ser Arg His Pro Asp Thr Leu
Leu Ala Ala Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg
atg ggc gaa aac ttt atc gct tct 576 Ser Pro Leu Val Ile Gly Leu Gly
Met Gly Glu Asn Phe Ile Ala Ser 180 185 190 gac cag ctg gcg ctg ttg
ccg gtg acc cgt cgc ttt atc ttc ctt gaa 624 Asp Gln Leu Ala Leu Leu
Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205 gag ggc gat att
gcg gaa atc act cgc cgt tcg gta aac atc ttc gat 672 Glu Gly Asp Ile
Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220 aaa act
ggc gcg gaa gta aaa cgt cag gat atc gaa tcc aat ctg caa 720 Lys Thr
Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln 225 230 235
240 tat gac gcg ggc gat aaa ggc att tac cgt cac tac atg cag aaa gag
768 Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu
245 250 255 atc tac gaa cag ccg aac gcg atc aaa aac acc ctt acc gga
cgc atc 816 Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly
Arg Ile 260 265 270 agc cac ggt cag gtt gat tta agc gag ctg gga ccg
aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro
Asn Ala Asp Glu 275 280 285 ctg ctg tcg aag gtt gag cat att cag atc
ctc gcc tgt ggt act tct 912 Leu Leu Ser Lys Val Glu His Ile Gln Ile
Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tcc ggt atg gtt tcc cgc
tac tgg ttt gaa tcg cta gca ggt 960 Tyr Asn Ser Gly Met Val Ser Arg
Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 att ccg tgc gac gtc
gaa atc gcc tct gaa ttc cgc tat cgc aaa tct 1008 Ile Pro Cys Asp
Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg
cgt cgt aac agc ctg atg atc acc ttg tca cag tct ggc gaa 1056 Ala
Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345
350 acc gcg gat acc ctg gct ggc ctg cgt ctg tcg aaa gag ctg ggt tac
1104 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly
Tyr 355 360 365 ctt ggt tca ctg gca atc tgt aac gtt ccg ggt tct tct
ctg gtg cgc 1152 Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser
Ser Leu Val Arg 370 375 380 gaa tcc gat ctg gcg cta atg acc aac gcg
ggt aca gaa atc ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn
Ala Gly Thr Glu Ile Gly Val 385 390 395 400 gca tcc act aaa gca ttc
acc act cag tta act gtg ctg ttg atg ctg 1248 Ala Ser Thr Lys Ala
Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag
ctg tct cgc ctg aaa ggt ctg gat gcc tcc att gaa cat 1296 Val Ala
Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430
gac atc gtg cat ggt ctg cag gcg ctg ccg agc cgt att gag cag atg
1344 Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln
Met 435 440 445 ctg tct cag gac aaa cgc att gaa gcg ctg gca gaa gat
ttc tct gac 1392 Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu
Asp Phe Ser Asp 450 455 460 aaa cat cac gcg ctg ttc ctg ggc cgt ggc
gat cag tac cca atc gcg 1440 Lys His His Ala Leu Phe Leu Gly Arg
Gly Asp Gln Tyr Pro Ile Ala 465 470 475 480 ctg gaa ggc gca ttg aag
ttg aaa gag atc tct tac att cac gct gaa 1488 Leu Glu Gly Ala Leu
Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495 gcc tac gct
gct ggc gaa ctg aaa cac ggt ccg ctg gcg cta att gat 1536 Ala Tyr
Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510
gcc gat atg ccg gtt att gtt gtt gca ccg aac aac gaa ttg ctg gaa
1584 Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu
Glu 515 520 525 aaa ctg aaa tcc aac att gaa gaa gtt cgc gcg cgt ggc
ggt cag ttg 1632 Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg
Gly Gly Gln Leu 530 535 540 tat gtc ttc gcc gat cag gat gcg ggt ttt
gta agt agc gat aac atg 1680 Tyr Val Phe Ala Asp Gln Asp Ala Gly
Phe Val Ser Ser Asp Asn Met 545 550 555 560 cac atc atc gag atg ccg
cat gtg gaa gag gtg att gca ccg atc ttc 1728 His Ile Ile Glu Met
Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575 tac acc gtt
ccg ctg cag ctg ctg gct tac cat gtc gcg ctg atc aaa 1776 Tyr Thr
Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590
ggc acc gac gtt gac cag ccg cgt aac ctg gca aaa tcg gtt acg gtt
1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr
Val 595 600 605 gag taa 1830 Glu <210> SEQ ID NO 9
<211> LENGTH: 609 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 9 Met Cys Gly Ile Val Gly
Ala Ile Ala Gln Arg Asp Val Ala Glu Ile 1 5 10 15 Leu Leu Glu Gly
Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Leu
Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45
Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50
55 60 His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly
Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His
Ile Val Val 85 90 95 Val His Asn Gly Ile Ile Glu Asn His Glu Pro
Leu Arg Glu Glu Leu 100 105 110 Lys Ala Arg Gly Tyr Thr Phe Val Ser
Glu Thr Asp Thr Glu Val Ile 115 120 125 Ala His Leu Val Asn Trp Glu
Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Ala Val Leu Arg Ala
Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 Ile Met
Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175
Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180
185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu
Glu 195 200 205 Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn
Ile Phe Asp 210 215 220 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile
Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Ala Gly Asp Lys Gly Ile
Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 Ile Tyr Glu Gln Pro Asn
Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270 Ser His Gly Gln
Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 Leu Leu
Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300
Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305
310 315 320 Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg
Lys Ser 325 330 335 Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser
Gln Ser Gly Glu 340 345 350 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu
Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala Ile Cys Asn
Val Pro Gly Ser Ser Leu Val Arg
370 375 380 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile
Gly Val 385 390 395 400 Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr
Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly
Leu Asp Ala Ser Ile Glu His 420 425 430 Asp Ile Val His Gly Leu Gln
Ala Leu Pro Ser Arg Ile Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys
Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His
Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala 465 470 475 480
Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485
490 495 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile
Asp 500 505 510 Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu
Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala
Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Ala Asp Gln Asp Ala Gly
Phe Val Ser Ser Asp Asn Met 545 550 555 560 His Ile Ile Glu Met Pro
His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575 Tyr Thr Val Pro
Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590 Gly Thr
Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605
Glu <210> SEQ ID NO 10 <211> LENGTH: 1830 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic sequence encoding
an Escherichia coli protein having the activity of a GFAT
<400> SEQUENCE: 10 atgtgcggaa ttgttggtgc tatcgcccaa
agagacgttg ctgagatttt gttagagggt 60 ctgcgaaggc tagagtatag
aggatatgac tccgctggtc tggctgtcgt tgatgctgag 120 ggtcatatga
caaggctaag aaggttagga aaggttcaga tgcttgctca ggcagctgag 180
gaacatccat tgcatggagg tactggtatt gcacatacca ggtgggctac tcatggggag
240 ccatcagaag ttaatgctca tccacatgtg agtgagcata tcgttgtagt
tcacaatggg 300 ataattgaaa accacgaacc attgagggaa gagttaaagg
caagaggata tacttttgtg 360 agtgagactg acactgaggt tattgcacat
ttagtgaact gggaactcaa acaggggggc 420 acattgcgtg aggctgtgtt
aagagctatt cctcaactta gaggtgcata cggtactgtt 480 attatggatt
caagacaccc agatactctc cttgcagcta gatcaggtag tcccttggtc 540
ataggacttg gaatgggtga aaattttatc gctagcgacc aattggcctt attgccagtt
600 acaagacgat ttattttcct tgaagagggc gatattgctg agattactag
aaggtctgtg 660 aacatctttg ataagactgg cgctgaggtt aaacgtcagg
atatcgagtc taaccttcaa 720 tacgatgctg gtgataaagg aatttacagg
cattatatgc aaaaggaaat ttatgaacaa 780 ccaaatgcta tcaaaaacac
acttactggc cgtatttctc atggacaggt cgatttaagc 840 gagcttggtc
ctaatgcaga cgaactgcta tcaaaagttg agcacataca gatactggca 900
tgcggaacta gttataattc aggaatggtc tctagatact ggttcgaaag cttggcaggt
960 ataccttgtg atgtagagat cgcttctgag tttaggtata gaaagtctgc
tgtgcgtaga 1020 aattcattaa tgattacatt atctcaatcc ggagaaacag
cagatacact ggctggattg 1080 aggctttcta aggaactcgg atatctgggt
tcacttgcta tttgtaatgt accaggttcc 1140 tcattggttc gtgaatcaga
tctagcactt atgacaaatg caggaactga aataggtgtg 1200 gcaagtacca
aggctttcac aacccaactg accgtacttt taatgttggt agcaaaactc 1260
agtcgattaa aggggctaga tgcatctatc gaacatgata ttgttcacgg gcttcaagct
1320 ctcccttcaa gaattgaaca aatgctttca caagataaga gaatagaggc
attggctgaa 1380 gatttttccg acaaacatca cgcattgttt cttggacgtg
gcgatcaata tccaattgca 1440 ttggaaggag ctttgaagtt gaaagaaata
agttacattc acgcagaagc atatgcagct 1500 ggagaactca agcatggtcc
tttggcactc atcgacgctg acatgcccgt gatcgtagtg 1560 gctcctaata
acgaactgct cgaaaagctt aaatcaaata tcgaagaggt tcgagctaga 1620
ggaggtcagc tttacgtttt cgctgaacaa gatgctggat tcgtgtcaag cgataatatg
1680 catataattg aaatgcctca cgttgaagaa gtgattgcac ctatatttta
tacagtccca 1740 ttgcaacttc tagcttacca tgttgcactt attaaaggaa
ctgatgttga tcagcctaga 1800 aacctagcaa aatctgtaac agtcgaataa 1830
<210> SEQ ID NO 11 <211> LENGTH: 1260 <212> TYPE:
DNA <213> ORGANISM: Paramecium bursaria Chlorella Virus 1
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (62)..(1228) <300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: U42580.4 <309>
DATABASE ENTRY DATE: 2004-09-20 <313> RELEVANT RESIDUES IN
SEQ ID NO: (291749.)..(292918) <400> SEQUENCE: 11 atcaacgtga
tttatatttt aaacaaagac cattcacatc tttagtactt aattaattat 60 a atg tca
cga atc gca gtc gtt ggt tgt ggt tac gtc gga acc gct tgt 109 Met Ser
Arg Ile Ala Val Val Gly Cys Gly Tyr Val Gly Thr Ala Cys 1 5 10 15
gca gta ctt ctt gct caa aaa aac gaa gtc atc gtg ctt gat att agc 157
Ala Val Leu Leu Ala Gln Lys Asn Glu Val Ile Val Leu Asp Ile Ser 20
25 30 gaa gac cgt gtt caa cta atc aag aac aag aag agt cca atc gag
gac 205 Glu Asp Arg Val Gln Leu Ile Lys Asn Lys Lys Ser Pro Ile Glu
Asp 35 40 45 aag gaa atc gaa gag ttt ctc gaa acg aaa gac ctg aac
ctg acc gcg 253 Lys Glu Ile Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn
Leu Thr Ala 50 55 60 acg act gac aag gtt ctt gca tac gaa aac gcc
gaa ttt gtc atc atc 301 Thr Thr Asp Lys Val Leu Ala Tyr Glu Asn Ala
Glu Phe Val Ile Ile 65 70 75 80 gca acc ccg act gac tat gac gtg gtt
act agg tat ttt aac acg aaa 349 Ala Thr Pro Thr Asp Tyr Asp Val Val
Thr Arg Tyr Phe Asn Thr Lys 85 90 95 tct gtg gaa aac gtc att ggg
gac gtg atc aaa aat aca cag acc cat 397 Ser Val Glu Asn Val Ile Gly
Asp Val Ile Lys Asn Thr Gln Thr His 100 105 110 cca act atc gtg att
aaa tct acc atc ccc att gga ttt gtt gat aag 445 Pro Thr Ile Val Ile
Lys Ser Thr Ile Pro Ile Gly Phe Val Asp Lys 115 120 125 gtt cgt gag
caa ttc gac tac caa aat atc att ttc tcc cca gaa ttt 493 Val Arg Glu
Gln Phe Asp Tyr Gln Asn Ile Ile Phe Ser Pro Glu Phe 130 135 140 ctg
cgt gaa ggt aga gcc ttg tat gat aat ctc tac cca tcc cgt atc 541 Leu
Arg Glu Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile 145 150
155 160 atc gta gga gat gat tcc ccc att gcg ctt aag ttc gca aac ctt
ctc 589 Ile Val Gly Asp Asp Ser Pro Ile Ala Leu Lys Phe Ala Asn Leu
Leu 165 170 175 gtt gaa ggt tct aaa act ccg ctt gcc cct gtc ctg acg
atg gga act 637 Val Glu Gly Ser Lys Thr Pro Leu Ala Pro Val Leu Thr
Met Gly Thr 180 185 190 cgc gaa gcc gag gcc gtc aaa cta ttc tct aac
acg tat ctt gca atg 685 Arg Glu Ala Glu Ala Val Lys Leu Phe Ser Asn
Thr Tyr Leu Ala Met 195 200 205 cga gtt gca tac ttc aac gaa cta gat
aca ttc gca atg tct cac ggt 733 Arg Val Ala Tyr Phe Asn Glu Leu Asp
Thr Phe Ala Met Ser His Gly 210 215 220 atg aat gcg aaa gaa atc att
gat ggt gtg act ctg gag cct cgc att 781 Met Asn Ala Lys Glu Ile Ile
Asp Gly Val Thr Leu Glu Pro Arg Ile 225 230 235 240 ggt cag ggg tac
tca aac cct tcg ttc ggt tat gga gct tat tgc ttt 829 Gly Gln Gly Tyr
Ser Asn Pro Ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255 cca aag
gat acg aag caa ctg ctg gct aat ttc gag gga gtg cct caa 877 Pro Lys
Asp Thr Lys Gln Leu Leu Ala Asn Phe Glu Gly Val Pro Gln 260 265 270
gat atc atc gga gca att gta gaa tca aat gag act cgc aag gaa gtg 925
Asp Ile Ile Gly Ala Ile Val Glu Ser Asn Glu Thr Arg Lys Glu Val 275
280 285 att gtg agt gaa gta gaa aat cgt ttc ccc acg act gtt ggt gtg
tat 973 Ile Val Ser Glu Val Glu Asn Arg Phe Pro Thr Thr Val Gly Val
Tyr 290 295 300 aag ctc gcc gct aaa gcg ggt tct gat aat ttt cgg agt
tct gca att 1021 Lys Leu Ala Ala Lys Ala Gly Ser Asp Asn Phe Arg
Ser Ser Ala Ile 305 310 315 320 gta gac ata atg gag cga ctt gca aac
aag ggt tat cac att aag att 1069 Val Asp Ile Met Glu Arg Leu Ala
Asn Lys Gly Tyr His Ile Lys Ile 325 330 335 ttc gaa cca act gtg gaa
caa ttc gaa aac ttt gaa gtt gat aac aac 1117 Phe Glu Pro Thr Val
Glu Gln Phe Glu Asn Phe Glu Val Asp Asn Asn 340 345 350 ctg aca aca
ttt gcg act gag agc gat gta att atc gca aac aga gtt 1165 Leu Thr
Thr Phe Ala Thr Glu Ser Asp Val Ile Ile Ala Asn Arg Val 355 360 365
ccc gtt gaa cat cgc att ctc ttt ggt aaa aaa tta atc aca cgt gat
1213 Pro Val Glu His Arg Ile Leu Phe Gly Lys Lys Leu Ile Thr Arg
Asp 370 375 380 gta tat ggc gat aac taaaatgttt tcaatatgat
gttgttaatg at 1260 Val Tyr Gly Asp Asn 385 <210> SEQ ID NO 12
<211> LENGTH: 389 <212> TYPE: PRT <213> ORGANISM:
Paramecium bursaria Chlorella Virus 1 <400> SEQUENCE: 12 Met
Ser Arg Ile Ala Val Val Gly Cys Gly Tyr Val Gly Thr Ala Cys 1 5 10
15 Ala Val Leu Leu Ala Gln Lys Asn Glu Val Ile Val Leu Asp Ile Ser
20 25 30 Glu Asp Arg Val Gln Leu Ile Lys Asn Lys Lys Ser Pro Ile
Glu Asp
35 40 45 Lys Glu Ile Glu Glu Phe Leu Glu Thr Lys Asp Leu Asn Leu
Thr Ala 50 55 60 Thr Thr Asp Lys Val Leu Ala Tyr Glu Asn Ala Glu
Phe Val Ile Ile 65 70 75 80 Ala Thr Pro Thr Asp Tyr Asp Val Val Thr
Arg Tyr Phe Asn Thr Lys 85 90 95 Ser Val Glu Asn Val Ile Gly Asp
Val Ile Lys Asn Thr Gln Thr His 100 105 110 Pro Thr Ile Val Ile Lys
Ser Thr Ile Pro Ile Gly Phe Val Asp Lys 115 120 125 Val Arg Glu Gln
Phe Asp Tyr Gln Asn Ile Ile Phe Ser Pro Glu Phe 130 135 140 Leu Arg
Glu Gly Arg Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile 145 150 155
160 Ile Val Gly Asp Asp Ser Pro Ile Ala Leu Lys Phe Ala Asn Leu Leu
165 170 175 Val Glu Gly Ser Lys Thr Pro Leu Ala Pro Val Leu Thr Met
Gly Thr 180 185 190 Arg Glu Ala Glu Ala Val Lys Leu Phe Ser Asn Thr
Tyr Leu Ala Met 195 200 205 Arg Val Ala Tyr Phe Asn Glu Leu Asp Thr
Phe Ala Met Ser His Gly 210 215 220 Met Asn Ala Lys Glu Ile Ile Asp
Gly Val Thr Leu Glu Pro Arg Ile 225 230 235 240 Gly Gln Gly Tyr Ser
Asn Pro Ser Phe Gly Tyr Gly Ala Tyr Cys Phe 245 250 255 Pro Lys Asp
Thr Lys Gln Leu Leu Ala Asn Phe Glu Gly Val Pro Gln 260 265 270 Asp
Ile Ile Gly Ala Ile Val Glu Ser Asn Glu Thr Arg Lys Glu Val 275 280
285 Ile Val Ser Glu Val Glu Asn Arg Phe Pro Thr Thr Val Gly Val Tyr
290 295 300 Lys Leu Ala Ala Lys Ala Gly Ser Asp Asn Phe Arg Ser Ser
Ala Ile 305 310 315 320 Val Asp Ile Met Glu Arg Leu Ala Asn Lys Gly
Tyr His Ile Lys Ile 325 330 335 Phe Glu Pro Thr Val Glu Gln Phe Glu
Asn Phe Glu Val Asp Asn Asn 340 345 350 Leu Thr Thr Phe Ala Thr Glu
Ser Asp Val Ile Ile Ala Asn Arg Val 355 360 365 Pro Val Glu His Arg
Ile Leu Phe Gly Lys Lys Leu Ile Thr Arg Asp 370 375 380 Val Tyr Gly
Asp Asn 385 <210> SEQ ID NO 13 <211> LENGTH: 1170
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
sequence encoding a Paramecium bursaria Chlorella Virus protein
having the activity of a UDP-Glc-DH <400> SEQUENCE: 13
atgtctcgca tagctgttgt aggatgtggc tatgtgggaa ctgcatgtgc ggttctactt
60 gctcaaaaga acgaagttat tgtgcttgat attagtgaag accgtgttca
acttattaag 120 aacaagaagt ctcctattga ggataaggaa atcgaagagt
tcttggaaac aaaggatctt 180 aatcttactg cgactacaga taaggttctt
gcctacgaga acgctgagtt tgtgataatc 240 gctacaccaa ccgattacga
cgttgtgact cgatatttca ataccaaatc cgtggaaaac 300 gttataggag
atgttatcaa gaacactcaa acccacccta ctatcgtcat caagtccaca 360
attcccatcg gtttcgttga taaggtcaga gagcagtttg attatcaaaa cattatcttc
420 tcacctgagt tcttaaggga gggtcgtgct ctctacgata atttgtatcc
gtcccgtatt 480 atcgttggcg acgattctcc tatcgctctc aagttcgcaa
atctcttagt tgagggtagt 540 aagacccctt tggctcctgt tttgacaatg
ggaaccagag aagcagaagc tgtcaagcta 600 ttctctaata cctaccttgc
catgagggta gcatacttta acgaacttga tacatttgct 660 atgtcgcatg
gtatgaatgc caaggagatt atagatggtg tcactttaga gcccaggatc 720
ggtcaaggat attctaaccc atcattcggc tatggagctt actgctttcc taaggacact
780 aagcagttgc tggcaaactt cgagggagtt cctcaagaca tcataggcgc
tattgtggag 840 tcaaacgaaa caaggaaaga ggtgatagtt agtgaggtag
agaatcgttt cccaacgaca 900 gtcggtgttt acaaactggc agctaaagct
ggtagcgata acttcaggtc aagtgctatt 960 gtcgacatca tggaacgcct
ggctaacaaa ggttaccaca ttaagatctt tgagccaact 1020 gtagagcagt
tcgaaaattt cgaagttgac aataacttga caacgtttgc tactgagtca 1080
gacgttatta tcgcaaatcg tgtccctgtg gaacatagaa tcctatttgg aaagaagctc
1140 attaccagag atgtttacgg tgataattaa 1170 <210> SEQ ID NO 14
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: synthetic oligonucleotide <400> SEQUENCE: 14
tcgacaggcc tggatcctta attaaactag tctcgaggag ctcggtac 48 <210>
SEQ ID NO 15 <211> LENGTH: 40 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: synthetic oligonucleotide
<400> SEQUENCE: 15 cgagctcctc gagactagtt taattaagga
tccaggcctg 40 <210> SEQ ID NO 16 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: synthetic
oligonucleotide <400> SEQUENCE: 16 aaaaactagt tctacatcgg
cttaggtgta gcaacacg 38 <210> SEQ ID NO 17 <211> LENGTH:
39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: synthetic
oligonucleotide <400> SEQUENCE: 17 aaaagatatc tgttgttgga
ttctactact atgcttcaa 39
* * * * *
References